Methods for treating bleeding disorders using a platelet subpopulation

ABSTRACT

The present invention relates to a platelet subpopulation with high binding capacity to recombinant activated factor VII (rFVIIa), and its use for the treatment of bleeding disorders and for determining whether a subject is a candidate for treatment with rFVIIa.

All patents, patent applications and publications, and non-patentpublications cited herein are hereby incorporated by reference in theirentirety. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/799,875, filed Mar. 15, 2013, the disclosure of which isincorporated by reference.

BACKGROUND OF THE INVENTION

The body can engage in blood clotting through the “extrinsic pathway”and the “intrinsic pathway.” Clotting factors mediate the clottingresponse in both pathways. Both pathways are activated by differentstimuli and ultimately feed into a common machinery. In the extrinsicpathway, when vascular injury occurs, a subendothelial cell-surfaceglycoprotein called “Tissue Factor” (TF) (also known as Factor III) isreleased at the site of injury triggering blood coagulation in healthyindividuals. Tissue factor is found on the outside of bloodvessels—normally not exposed to the bloodstream.

Coagulation is mainly triggered by FVIIa activating FX after exposure ofTF at the broken vessel wall. Approximately one percent of FVII iscirculating in its activated enzyme form (FVIIa). FVIIa forms theso-called “extrinsic tenase” complex with TF leading to activation offactor X (FX) to FXa, which leads to activation of initial levels ofthrombin, and to activation of FIX to FIXa. Thrombin is required toactivate FVIII, FV, and platelets. Formation of the “intrinsic tenase”complex consisting of FIXa and FVIIIa leads to activation of FX at arate which is several orders of magnitude higher than by the FVIIa/TFcomplex. These amounts of FXa are required to cause the “thrombin burst”leading to formation of enough fibrin to form a stable blood clot. Allthese processes occur on the membrane surfaces of activated platelets.

Platelets are anucleic cells that circulate in the blood of mammals. Inthe absence of trauma, the inner surface of blood vessels is lined witha thin layer of endothelial cells that acts to inhibit plateletactivation. When blood vessels are damaged, fibrils of collagen in theextracellular matrix (ECM) are exposed. Platelets then begin to adhereto the collagen through the action of specific receptors for collagenpresent on their plasma membrane. These adhesions activate the plateletsin addition to the earlier described mechanisms. Platelets are alsoactivated by thrombin after initiation of coagulation.

Hemophilia refers to a group of bleeding disorders in which it takes along time for the blood to clot. Hemophilia A is the most common form ofthe disorder and is caused by a deficiency in Factor VIII. Hemophilia Bis less frequent and is caused by a deficiency in Factor IX. Both formsof hemophilia can be effectively treated by administration of eitherrecombinant or plasma derived FVIII or FIX concentrates. Treatment ofhemophilia is complicated by the development of inhibitory antibodies tofactors VIII or IX. In the case of Factor VIII (FVIII), inhibitorsdevelop in ˜30% of the patients. Currently approved therapies in thesecases include the infusion of plasma-derived prothrombin complexconcentrates, like FEIBA, or recombinant Factor VIIa (rFVIIa), as thetherapies for acute bleeds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. FACS analyses of non-activated platelets with 100 nM rFVIIa(Novoseven) added for 7 minutes at 37° C. using CD61 PerCP, CD62 PE, andFVII DyLight 488 as markers.

FIG. 2. FACS analyses of platelets activated for 7 minutes at 37° C.with thrombin/convulxin and with 100 nM NovoSeven added using CD61PerCP, CD62 PE, and FVII DyLight 488 as markers.

FIG. 3. Histogram overlay of the CD61PerCP staining for differentsamples as indicated. Population is gated by forward scatter versus sidescatter. Graph showing detection of platelets using CD61 (GPIIIa,Integrin b3) as total platelet marker. Shown are three histograms eachof dual-agonist activated and non activated platelet samples with rFVIIaadded and stained with a mouse monoclonal antibody against human CD61 orwith an IgG1 isotype control with the same fluorochrome label PerCP. Theisotype control (black solid line) was also an activated sample.Activation led to a shift of peaks towards a higher signal.

FIG. 4. Histogram overlay of the CD62PE staining for different samplesas indicated. Population is gated by forward scatter versus side scatterand CD61. Graph showing measurement of platelet activation using CD62P(P-selectin) as marker for activated platelets. One histogram (blackline) represents non-activated platelets with rFVIIa added and incubatedfor 10 min. Three histograms show platelets after dual-agonistactivation with thrombin and convulxin in presence of 100 nM rFVIIaafter 7 minutes (red line), 10 minutes (green line), and 15 min (blueline) incubation time.

FIG. 5. CD62P signal as an indicator for platelet activation at a rangeof rFVIIa concentrations from 100 to 2000 nM. The CD62P signal wascompared to non-activated platelet background in Novoseven N7 or BAX817formulation buffer. Shown are CD62P signal to noise ratios ofnon-activated or dual-agonist activated platelets with 100-2000 nMrFVIIa bound (Novoseven or BAX817).

FIGS. 6A-B. Titration of rFVIIa (BAX=BAX817, N7=Novoseven) on platelets.The median fluorescence values of rFVIIa bound to activated andnon-activated platelets (CD61-positive population) are shown. FIG. 6A.Ratio of binding of rFVIIa to activated vs. non-activated platelets.FIG. 6B. Fluorescence signal to noise ratios of rFVIIa bound toactivated or non-activated platelets.

FIG. 7. Histogram overlay of the FVII DyLight488 staining for plateletsafter different activation times with thrombin/convulxin in presence orabsence of 100 nM rFVIIa (Novoseven) as indicated. Populations are gatedby forward scatter versus side scatter and CD61.

FIG. 8. FVIIa staining on non-activated (upper panel and dottedhistogram lines) and thrombin and convulxin dual agonist activated(lower panel and solid histogram lines) platelets. Platelets wereidentified by forward- and side-scatter distribution and CD61counterstaining. Stained platelets treated without rFVIIa are shown onthe left side, platelets incubated with 100 nM rFVIIa on the right.

FIG. 9. FVIIa and CD62P or Fibrinogen counterstaining on non-activated(upper panel) and thrombin and convulxin dual agonist activated (lowerpanel) platelets. CD62P surface expression versus rFVIIa binding ofplatelets treated without rFVIIa is shown on the left side, CD62Psurface expression versus FVIIa binding of platelets incubated with 100nM rFVIIa BAX817 is shown in the middle. Fibrinogen surface expressionversus FVIIa binding of platelets treated with 100 nM rFVIIa fromNovoSeven (NS) is shown on the right. CD62P expression on resting oractivated platelets treated with of 100 nM NovoSeven and 100 nM BAX817was comparable.

FIG. 10. FACS analysis showing platelet characteristics. The left-handpanel shows a non-activated platelet population and rFVIIa. Theright-hand panel shows an activated platelet population and rFVIIa.

FIG. 11. FACS analysis showing a time course of the rFVIIa signal versusthe CD26P signal on activated platelets.

FIG. 12. Dot plot of FVII versus CD62P of Novoseven lot#1 bound todifferent platelet samples as indicated. Platelets with 100 nM Novosevenwere thrombin/convulxin activated for 7 minutes (top), 10 minutes(middle), or 15 minutes (bottom). Platelets were identified viaforward/side scatter properties and CD61 staining. Percentages of theCD61-population falling into the two regions defining the major andhigh-rFVIIa binding populations are shown just above each region.

FIG. 13. Dot plot of FVII versus CD62P of Novo seven lot#2 bound todifferent platelet samples as indicated. Platelets with 100 nM Novosevenwere thrombin/convulxin activated for 7 minutes (top), 10 minutes(middle), or 15 minutes (bottom). Platelets were identified viaforward/side scatter properties and CD61 staining. Percentages of theCD61-population falling into the two regions defining the major andhigh-rFVIIa binding populations are given.

FIG. 14. Histogram showing the statistics of the time course of therFVIIa signal on activated platelets.

FIG. 15. Dotplots of BAX817 lots and N7 gated on CD61. 100 nM rFVIIa anda 7 min dual agonist activation were used. Panels A-E use the BAX817lots; panels F-I use N7; panel 0) uses Des-GlaFVIIa; panel e) uses theBAX817 lots; and panel h) uses N7 lot on non-activated platelets.

FIG. 16A-B. Charts providing a statistical overview of plateletpopulations and high rFVIIa binding subpopulations from 37 donors. FIG.16A summarizes analysis of non-activated platelets. FIG. 16B summarizesanalysis of dual-agonist activated platelets. Percentages of the highrFVIIa binding subpopulation of all platelets, the approximateconcentration when saturation was reached, ratios of median fluorescenceintensities (MFIs) of the high binding versus the main plateletspopulation (at all rFVIIa concentrations measured and for 100 nMrFVIIa), and relative fluorescence intensities (X-fold MFI,MFI_(sample)/MFI_(control)) of rFVIIa bound to all platelets compared toa control population not exposed to rFVIIa (at all rFVIIa concentrationsmeasured and for 100 nM rFVIIa) are provided. Analysis includes X-foldMFI of high capacity binders versus the major population when the highrFVIIa binding population was at least 2% of total platelets.

FIG. 17A-B. Population sizes and median fluorescence intensities (MFIs)for rFVIIa and fibrinogen for non-activated (FIG. 17A) and dual-agonistactivated (FIG. 17B) platelet samples from five donors. rFVIIa andfibrinogen bound to platelets defined subpopulations, which wereseparated by “spidery” quadrant gates. Percentages of each population ofall platelets and median fluorescence intensity (MFIs) of the FVII/FVIIaand fibrinogen staining are provided. If no rFVIIa was added, quadrants(Q) show the following: Q1: FVIIa negative, Fibrinogen positive; Q2:FVIIa positive, Fibrinogen positive; Q3: FVIIa positive, Fibrinogennegative; Q4: FVIIa negative, Fibrinogen negative. For all othersamples, quadrants correspond to Q1: FVIIa main, Fibrinogen positive;Q2: FVIIa high, Fibrinogen positive; Q3: FVIIa high, Fibrinogennegative; Q4: FVIIa main, Fibrinogen negative. n/a: no events weredetected in that gate and thus no MFI is reported.

FIG. 18A-B. Bubble diagrams displaying median fluorescence intensities(MFIs) and percentages of platelet subpopulations identified by rFVIIaand fibrinogen staining for dual-agonist activated platelet samples fromdifferent donors. Using spider quadrant gates, four main populationswere separated and were described by their rFVIIa staining intensity onthe x-axis, fibrinogen staining on the y-axis, and relative size (sizeof bubble corresponds to percentage of all platelets in the sample).Detailed raw data and quadrant statistics for all donors are provided inFIGS. 17A-B. rFVIIa and fibrinogen staining revealed differentsubpopulation sizes in most donors tested. FIG. 18A illustratesdistributions of activated but not coated, and activated and coatedplatelets in four different donors with no rFVIIa added. A small portionof platelets may contain endogenous FVII/FVIIa. FIG. 18B illustratesdistributions of rFVIIa binding populations detected after addition of100 nM rFVIIa.

FIG. 19. Boxplot diagram illustrating the distribution of rFVIIa bindingcapacity increases observed in platelet samples from 37 donors. Theratio of median fluorescence intensity (corresponding to rFVIIa binding)of dual-agonist treated (activated) platelets versus non-treated(non-activated) platelets is provided on the y-axis. Platelets fromdonors having the least rFVIIa binding activity (the bottom 10% ofdonors) bound less than 1.3-fold more rFVIIa upon dual-agonist treatmentcompared with binding activity of non-activated platelets. Plateletsfrom the bottom 25% of donors did not demonstrate an increase of morethan 1.6-fold binding of rFVIIa upon dual-agonist activation.Dual-agonist activated platelets from donors having the highest level ofbinding activity (the top 10% of donors) bound more than 3.5-fold rFVIIacompared with non-activated platelets. The top 25% of donors hadplatelets that bound more than 2.2-fold rFVIIa upon dual-agonistactivation.

FIG. 20A-B. Boxplot diagrams illustrating the distribution of overallcapacity of non-stimulated (FIG. 20A) and dual-agonist activated (FIG.20B) platelets to bind rFVIIa upon treatment with 100 nM rFVIIa. Thefold increase of median fluorescence intensity (corresponding to rFVIIabinding) compared to a buffer control is provided on the y-axis. Theline bisecting the shaded box indicates that 50% of donors have anX-fold MFI lower or higher than this value. The borders of the shadedbox indicate that top and bottom 25% of the population have the X-foldMFI higher or lower, respectively, of the indicated value. The terminiof the perpendicular lines rising from the shaded box (the “whisker”)indicate the X-fold MFI for the top and bottom 10% of the donorpopulation.

SUMMARY OF THE INVENTION

The present invention relates to a platelet subpopulation with highbinding capacity to recombinant activated factor VII (rFVIIa), and itsuse for the treatment of bleeding disorders. The present invention alsorelates to methods for determining whether a subject is a candidate fortreatment with rFVIIa or alternative therapies.

In one aspect, the present invention provides a method of treatinghemophilia in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of rFVIIa to thesubject, wherein (a) a sample of platelets derived from the subject wasobtained; (b) a platelet population from the sample was incubated withrFVIIa; and (c) a subpopulation of platelets having at least a 4-foldhigher binding capacity to rFVIIa, as compared to the binding capacityto rFVIIa of the other platelets in the platelet population, wasdetected in the platelet population.

In another aspect, the present invention provides a method of treatinghemophilia in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an alternativetherapy to the subject, wherein (a) a sample of platelets derived fromthe subject was obtained; (b) a platelet population from the sample wasincubated with rFVIIa; and (c) a subpopulation of platelets having atleast a 4-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation, was not detected in the platelet population.

In another aspect, the present invention provides a method of treating anon-hemophilia bleeding disorder in a subject in need thereof, themethod comprising administering a therapeutically effective amount ofrFVIIa to the subject, wherein (a) a sample of platelets derived fromthe subject was obtained; (b) a platelet population from the sample wasincubated with rFVIIa; (c) a subpopulation of platelets having at leasta 4-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of the other platelets in the platelet population,was detected in the platelet population; and (d) the subject does nothave an unacceptable risk of thrombosis.

In another aspect, the present invention provides a method of treating anon-hemophilia bleeding disorder in a subject in need thereof, themethod comprising administering a therapeutically effective amount of analternative therapy to the subject, wherein (a) a sample of plateletsderived from the subject was obtained; (b) a platelet population fromthe sample was incubated with rFVIIa; and (c) a subpopulation ofplatelets having at least a 4-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population, was not detected in the platelet population.

In yet another aspect, the invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of aplatelet population enriched with a subpopulation of platelets having atleast a 4-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation.

In one aspect, the present invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of rFVIIa to thesubject, wherein (a) a sample of platelets derived from the subject wasobtained; (b) a platelet population from the sample was incubated withrFVIIa; and (c) a subpopulation of platelets having at least a 4-foldhigher binding capacity to rFVIIa, as compared to the binding capacityto rFVIIa of the other platelets in the platelet population, wasdetected in the platelet population.

In yet another aspect, the present invention provides a method oftreating a bleeding disorder in a subject in need thereof, the methodcomprising administering a therapeutically effective amount of analternative therapy to the subject, wherein (a) a sample of plateletsderived from the subject was obtained; (b) a platelet population fromthe sample was incubated with rFVIIa; and (c) a subpopulation ofplatelets having at least a 4-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population, was not detected in the platelet population.

In one embodiment, the detection is by flow cytometry. In oneembodiment, the platelets in the subpopulation of platelets areactivated. In another embodiment, the platelets in the subpopulation ofplatelets are non-activated. In one embodiment, the platelets in thesubpopulation of platelets are coated.

In one embodiment, the platelet population contains a subpopulation ofplatelets having about a 6-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population. In another embodiment, the platelet populationcontains a subpopulation of platelets having about a 20-fold higherbinding capacity to rFVIIa, as compared to the binding capacity torFVIIa of the other platelets in the platelet population. In anotherembodiment, the platelet population contains a subpopulation ofplatelets having about a 30-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population. In one embodiment, the platelet population containsa subpopulation of platelets having about a 40-fold higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of theother platelets in the platelet population.

In one embodiment, the sample of platelets is obtained from a bloodsample or a serum sample from the subject. In one embodiment, the sampleof platelets is a fresh sample, a concentrate, a preserved sample, arehydrated lyophilized sample, or a frozen sample.

In one embodiment, the bleeding disorder is hemophilia. In oneembodiment, the hemophilia is hemophilia A. In another embodiment, thehemophilia is hemophilia B. In one embodiment, the hemophilia A iscongenital hemophilia A with inhibitors or acquired hemophilia A withinhibitory auto antibodies to FVIII, and the hemophilia B is congenitalhemophilia B with inhibitors or acquired hemophilia B with inhibitoryauto antibodies to FIX.

In one embodiment, the subject does not have an unacceptable risk ofthrombosis. In another embodiment, the subject has an unacceptable riskof thrombosis.

In one embodiment, the alternative therapy is Prothrombin ComplexConcentrate or activated Prothrombin Complex Concentrate. In oneembodiment, the activated Prothrombin Complex Concentrate is FEIBA.

In one embodiment, the alternative therapy is BeneFix®, Kogenate® FS,Recombinate, Advate®, Helixate® FS, Koāte®-DVI, Stimate®, DDAVP®,Bebulin, Hemofil M®, cryoprecipitated antihaemophilic factor (AHF), orfresh frozen plasma (FFP).

In another embodiment, the alternative therapy is recombinant porcineFVIII, recombinant FV variants, recombinant FVIIa variants, recombinantFXa variants, FXIII, prothrombin, fibrinogen, a mix of coagulationfactors, antibodies mimicking FVIII, peptides mimicking FVIII, compoundsmimicking FVIII, peptide inhibitors of TFPI, antibody inhibitors ofTFPI, compounds inhibiting TFPI or compounds inhibiting anti-coagulantproteins.

In one embodiment, the bleeding disorder is a non-hemophilia bleedingdisorder. In one embodiment, the non-hemophilia bleeding disorder isblood loss from trauma, FVII deficiency, FV deficiency, FX deficiency,FXI deficiency, FXIII deficiency, fibrinogen deficiency, prothrombindeficiency, dilutional coagulopathy, thrombocytopenia, blood loss fromhigh-risk surgeries, intracerebral hemorrhage, von Willebrand disease orvon Willebrand disease with inhibitors to von Willebrand factor.

In one embodiment, the bleeding disorder is hemophilia, blood loss fromtrauma, FVII deficiency, FV deficiency, FX deficiency, FXI deficiency,FXIII deficiency, fibrinogen deficiency, prothrombin deficiency,dilutional coagulopathy, thrombocytopenia, blood loss from high-risksurgeries, intracerebral hemorrhage, von Willebrand disease or vonWillebrand disease with inhibitors to von Willebrand factor.

In another aspect, the present invention provides a subpopulation ofplatelets isolated from a platelet population, wherein the platelets inthe subpopulation of platelets have at least a 4-fold higher bindingcapacity to rFVIIa as compared to the binding capacity to rFVIIa of theother platelets in the platelet population.

In one embodiment, the higher binding capacity to rFVIIa is determinedby flow cytometry. In one embodiment, the platelets in the subpopulationof platelets are activated. In another embodiment, the platelets in thesubpopulation of platelets are non-activated. In another embodiment, theplatelets in the subpopulation of platelets are coated.

In one embodiment, the platelets in the subpopulation of platelets haveabout a 6-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation. In another embodiment, the platelets in the subpopulation ofplatelets have about a 20-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population. In one embodiment, the platelets in thesubpopulation of platelets have about a 30-fold higher binding capacityto rFVIIa, as compared to the binding capacity to rFVIIa of the otherplatelets in the platelet population. In another embodiment, theplatelets in the subpopulation of platelets have about a 40-fold higherbinding capacity to rFVIIa, as compared to the binding capacity torFVIIa of the other platelets in the platelet population.

In one embodiment, the subpopulation of platelets is supplied in theform of a pharmaceutical composition. In one embodiment, thepharmaceutical composition is administered systemically to a subject inneed thereof.

In one embodiment, the pharmaceutical composition is used to treat ableeding disorder. In one embodiment, the bleeding disorder ishemophilia, blood loss from trauma, FVII deficiency, FV deficiency, FXdeficiency, FXI deficiency, FXIII deficiency, fibrinogen deficiency,prothrombin deficiency, dilutional coagulopathy, thrombocytopenia, bloodloss from high-risk surgeries, intracerebral hemorrhage, von Willebranddisease, or von Willebrand disease with inhibitors to von Willebrandfactor.

In another aspect, the present invention provides a method ofdetermining whether a subject is a candidate for treatment with rFVIIa,the method comprising (a) obtaining a sample of platelets derived fromthe subject; (b) incubating a platelet population from the sample withrFVIIa; (c) detecting whether the platelet population contains asubpopulation of platelets having at least a 4-fold higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of theother platelets in the platelet population, wherein a subject is acandidate for treatment with rFVIIa if the subject does not have anunacceptable risk of thrombosis, and if the platelet population containsa subpopulation of platelets having at least a 4-fold higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of theother platelets in the platelet population.

In one embodiment, the detection is by flow cytometry. In oneembodiment, the platelets in the subpopulation of platelets areactivated. In another embodiment, the platelets in the subpopulation ofplatelets are non-activated. In one embodiment, the platelets in thesubpopulation of platelets are coated.

In one embodiment, the platelet population contains a subpopulation ofplatelets having about a 6-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population. In another embodiment, the platelet populationcontains a subpopulation of platelets having about a 20-fold higherbinding capacity to rFVIIa, as compared to the binding capacity torFVIIa of the other platelets in the platelet population. In anotherembodiment, the platelet population contains a subpopulation ofplatelets having about a 30-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population. In one embodiment, the platelet population containsa subpopulation of platelets having about a 40-fold higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of theother platelets in the platelet population.

In one embodiment, the sample of platelets is obtained from a bloodsample or a serum sample from the subject. In another embodiment, thesample of platelets is a fresh sample, a concentrate, a preservedsample, a rehydrated lyophilized sample, or a frozen sample.

In one embodiment, the subject has a bleeding disorder. In oneembodiment, the bleeding disorder is hemophilia, blood loss from trauma,FVII deficiency, FV deficiency, FX deficiency, FXI deficiency, FXIIIdeficiency, fibrinogen deficiency, prothrombin deficiency, dilutionalcoagulopathy, thrombocytopenia, blood loss from high-risk surgeries,intracerebral hemorrhage, von Willebrand disease or von Willebranddisease with inhibitors to von Willebrand factor. In another embodiment,the bleeding disorder is blood loss from trauma, FVII deficiency, FVdeficiency, FX deficiency, FXI deficiency, FXIII deficiency, fibrinogendeficiency, prothrombin deficiency, dilutional coagulopathy,thrombocytopenia, blood loss from high-risk surgeries, intracerebralhemorrhage, von Willebrand disease or von Willebrand disease withinhibitors to von Willebrand factor.

In another aspect, the present invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of aplatelet population, wherein the platelets in the platelet populationhave a higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of platelets in a control platelet population.

In one embodiment, the platelets in the population of platelets areactivated. In another embodiment, the platelets in the population ofplatelets are non-activated. In one embodiment, the platelets in thepopulation of platelets are coated.

In one embodiment, the platelets in the platelet population have about a2-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of platelets in a control platelet population. In oneembodiment, the platelets in the platelet population have about a 5-foldhigher binding capacity to rFVIIa, as compared to the binding capacityto rFVIIa of platelets in a control platelet population. In oneembodiment, the platelets in the platelet population have about a10-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of platelets in a control platelet population. Inanother embodiment, the platelets in the platelet population have abouta 20-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of platelets in a control platelet population. In oneembodiment, the platelets in the platelet population have about a30-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of platelets in a control platelet population. Inanother embodiment, the platelets in the platelet population have abouta 40-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of platelets in a control platelet population.

In one embodiment, the platelets in the control platelet population areplatelets not exposed to rFVIIa. In one embodiment, the platelets in theplatelet population have a rFVIIa binding constant of about 50 to 400nM. In another embodiment, the platelets in the platelet population havea rFVIIa binding constant of about 25 to 1100 nM.

In one embodiment, the bleeding disorder is hemophilia, blood loss fromtrauma, FVII deficiency, FV deficiency, FX deficiency, FXI deficiency,FXIII deficiency, fibrinogen deficiency, prothrombin deficiency,dilutional coagulopathy, thrombocytopenia, blood loss from high-risksurgeries, intracerebral hemorrhage, von Willebrand disease or vonWillebrand disease with inhibitors to von Willebrand factor.

In one embodiment, the subject is a human.

In another aspect, the present invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of rFVIIa to thesubject, wherein (a) a sample of platelets derived from the subject wasobtained; (b) a platelet population from the sample was incubated withrFVIIa; and (c) platelets having a high binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of a control plateletpopulation, were detected in the platelet population.

In another aspect, the present invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an alternativetherapy to the subject, wherein (a) a sample of platelets derived fromthe subject was obtained; (b) a platelet population from the sample wasincubated with rFVIIa; and (c) platelets having a high binding capacityto rFVIIa, as compared to the binding capacity to rFVIIa of a controlplatelet population, were detected in the platelet population.

In another aspect, the present invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an alternativetherapy to the subject, wherein (a) a sample of platelets derived fromthe subject was obtained; (b) a platelet population from the sample wasincubated with rFVIIa; and (c) platelets having a low binding capacityto rFVIIa, as compared to the binding capacity to rFVIIa of a controlplatelet population, were detected in the platelet population.

In one embodiment, the detection is by flow cytometry. In oneembodiment, the platelets are activated. In another embodiment, theplatelets are non-activated. In one embodiment, the platelets arecoated.

In one embodiment, the control platelet population is a plateletpopulation not exposed to rFVIIa.

In one embodiment the control platelet population has a rFVIIa bindingconstant of about 200 nM.

In one embodiment, the platelets having a high binding capacity torFVIIa have about a 25-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of a control plateletpopulation. In another embodiment, the platelets having a high bindingcapacity to rFVIIa have about a 35-fold higher binding capacity torFVIIa, as compared to the binding capacity to rFVIIa of a controlplatelet population.

In one embodiment, the platelets having a low binding capacity to rFVIIahave about a 5-fold higher binding capacity to rFVIIa, as compared tothe binding capacity to rFVIIa of a control platelet population. Inanother embodiment, the platelets having a low binding capacity torFVIIa have about a 10-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of a control plateletpopulation.

In one embodiment, the sample of platelets is obtained from a bloodsample or a serum sample from the subject. In one embodiment, the sampleof platelets is a fresh sample, a concentrate, a preserved sample, arehydrated lyophilized sample, or a frozen sample.

In one embodiment, the bleeding disorder is hemophilia. In oneembodiment, the hemophilia is hemophilia A or hemophilia B. In anotherembodiment, the hemophilia A is congenital hemophilia A with inhibitorsor acquired hemophilia A with inhibitory auto antibodies to FVIII, andthe hemophilia B is congenital hemophilia B with inhibitors or acquiredhemophilia B with inhibitory auto antibodies to FIX.

In one embodiment, the subject does not have an unacceptable risk ofthrombosis. In another embodiment, the subject has an unacceptable riskof thrombosis.

In one embodiment, the bleeding disorder is a non-hemophilia bleedingdisorder. In one embodiment, the non-hemophilia bleeding disorder isblood loss from trauma, FVII deficiency, FV deficiency, FX deficiency,FXI deficiency, FXIII deficiency, fibrinogen deficiency, prothrombindeficiency, dilutional coagulopathy, thrombocytopenia, blood loss fromhigh-risk surgeries, intracerebral hemorrhage, von Willebrand disease orvon Willebrand disease with inhibitors to von Willebrand factor.

In one embodiment, the alternative therapy is Prothrombin ComplexConcentrate or activated Prothrombin Complex Concentrate. In oneembodiment, the activated Prothrombin Complex Concentrate is FEIBA.

In one embodiment, the alternative therapy is BeneFix®, Kogenate® FS,Recombinate, Advate®, Helixate® FS, Koāte®-DVI, Stimate®, DDAVP®,Bebulin, Hemofil M®, cryoprecipitated antihaemophilic factor (AHF), orfresh frozen plasma (FFP).

In another embodiment, the alternative therapy is recombinant porcineFVIII, recombinant FV variants, recombinant FVIIa variants, recombinantFXa variants, FXIII, prothrombin, fibrinogen, a mix of coagulationfactors, antibodies mimicking FVIII, peptides mimicking FVIII, compoundsmimicking FVIII, peptide inhibitors of TFPI, antibody inhibitors ofTFPI, compounds inhibiting TFPI or compounds inhibiting anti-coagulantproteins.

In one embodiment, the subject is a human.

The invention further provides a method of treating a bleeding disorderin a subject in need thereof. The method comprises administering to thesubject a therapeutically effective amount of a platelet population,wherein platelets in the platelet population demonstrate at least a15-fold higher rFVIIa relative fluorescence after exposure to 100 nMrFVIIa. In various embodiments, platelet population comprises (i)non-activated platelets demonstrating at least a 25-fold higher rFVIIarelative fluorescence after exposure to 100 nM rFVIIa and/or (ii)activated platelets demonstrating at least a 20-fold higher rFVIIarelative fluorescence (e.g., a least a 30-fold higher rFVIIa relativefluorescence) after exposure to 100 nM rFVIIa.

Also provided is a method of treating a bleeding disorder in a subjectin need thereof, the method comprising administering a therapeuticallyeffective amount of rFVIIa to the subject, wherein a) a sample ofplatelets derived from the subject was obtained; b) a non-activatedplatelet population from the sample was incubated with rFVIIa; and c)platelets demonstrating at least a 15-fold higher, optionally at least a25-fold higher, rFVIIa relative fluorescence after exposure to 100 nMrFVIIa were detected in the platelet population. Alternatively, themethod comprises administering a therapeutically effective amount ofrFVIIa to the subject, wherein a) a sample of platelets derived from thesubject was obtained; b) an activated platelet population from thesample was incubated with rFVIIa; and c) platelets demonstrating atleast a 20-fold higher, optionally at least a 30-fold higher, rFVIIarelative fluorescence after exposure to 100 nM rFVIIa were detected inthe platelet population. The invention also provides a method oftreating a bleeding disorder in a subject in need thereof, the methodcomprising administering a therapeutically effective amount of analternative therapy to the subject, wherein a) a sample of plateletsderived from the subject was obtained; b) a non-activated plateletpopulation from the sample was incubated with rFVIIa; and c) plateletsdemonstrating an 8-fold higher rFVIIa relative fluorescence or less,optionally a 5-fold higher rFVIIa relative fluorescence or less, afterexposure to 100 nM rFVIIa were detected in the platelet population.Alternatively, the method comprises administering a therapeuticallyeffective amount of an alternative therapy to the subject, wherein a) asample of platelets derived from the subject was obtained; b) anactivated platelet population from the sample was incubated with rFVIIa;and c) platelets demonstrating a 9-fold higher rFVIIa relativefluorescence or less, optionally a 5-fold higher rFVIIa relativefluorescence or less, after exposure to 100 nM rFVIIa were detected inthe platelet population.

A method of treating a bleeding disorder in a subject in need thereofalso is contemplated, wherein the method comprises administering atherapeutically effective amount of rFVIIa to a subject having plateletsthat demonstrate at least a 3.5-fold increase in binding capacity upondual-agonist activation compared to rFVIIa binding capacity ofnon-activated platelets from the subject. A method of treating ableeding disorder in a subject in need thereof is further providedwherein the method comprises administering a therapeutically effectiveamount of an alternative therapy to a subject having platelets thatdemonstrate a 1.3-fold increase in binding capacity or less upondual-agonist activation compared to rFVIIa binding capacity ofnon-activated platelets from the subject.

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description. The entire document is intended to berelated as a unified disclosure, and it should be understood that allcombinations of features described herein are contemplated, even if thecombination of features are not found together in the same sentence, orparagraph, or section of this document. For example, features of theinvention described with respect to a method of treating a bleedingdisorder also apply to methods of determining whether a subject is acandidate for rFVIIa treatment, and vice versa. Similarly, features ofthe invention described with respect to an isolated plateletsubpopulation also apply to methods of treating a bleeding disorder or amethod of determining whether a subject is a candidate for rFVIIatreatment.

DETAILED DESCRIPTION Definitions and Abbreviations

The term “N7” designates the drugs Novoseven® and Novoseven® RT.Novoseven® and Novoseven® RT are recombinant human Factor VIIa (rFVIIa),intended for promoting hemostasis by activating the extrinsic pathway ofthe coagulation cascade. Dosage and methods of administration ofNovoseven® and Novoseven® RT are known to one of skill in the art. Formore information, see, e.g., www.novosevenrt.com/.

The term “BAX817” designates the drug BAX817. BAX817 is recombinantFactor VIIa therapy, intended for treating acute bleedings in subjectswith hemophilia A or B with Factor VIII or Factor IX inhibitors.

The term “rFVIIa” designates recombinant Factor VIIa. Indications,dosage and methods of administration of rFVIIa are known to one of skillin the art (see e.g., Ng and Lee, 2006, Vasc Health Risk Manag., 2(4):433-440; see also Abshire and Kenet, 2004, J Thromb Haemost., 2(6):899-909.)

The term “non-hemophilia bleeding disorder” designates a bleedingdisorder that is not hemophilia (e.g., not hemophilia A, hemophilia B,hemophilia A with inhibitors, hemophilia B with inhibitors).

An “unacceptable risk of thrombosis” is a risk that is not medicallyappropriate in view of the potential benefit to the patient.

The term “coated,” as applied to coated platelets, represents asubpopulation of cells observed after dual agonist stimulation ofplatelets with collagen (or a collagen receptor agonist (e.g.,convulxin)) and thrombin. These platelets retain on their surface highlevels of several procoagulant proteins, including fibrinogen, vonWillebrand factor, fibronectin, factor V and thrombospondin. Foradditional information, see e.g., Dale G L., 2005, J Thromb Haemost.(10):2185-92. rFVIIa has been shown to preferentially bind to the“coated” platelet population emerging after activation with thrombin andconvulxin, a GPVI agonist (Kjalke M., 2007, J Thromb Haemost.(5):774-80.). It has also been shown that a “coated” plateletsub-population exposes high levels of Factor V, fibrinogen/fibrin, vWF(von Willebrand Factor), fibronectin, negatively charged phospholipids(see Kjalke et al., JTH 2007; 5: 774-80). In various embodiments,platelets that are not coated are activated platelets lacking fibrinogenon the platelet surface (or lacking more fibrinogen than naturally foundon non-activated platelets).

A “control platelet population” is, in various aspects, a plateletpopulation that is not exposed to rFVIIa.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. The term “or” should beunderstood to encompass items in the alternative or together, unlesscontext unambiguously requires otherwise. If aspects of the inventionare described as “comprising” a feature, embodiments also arecontemplated “consisting of” or “consisting essentially of” the feature.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

DETAILED DESCRIPTION

The invention relates to a platelet subpopulation with high bindingcapacity to recombinant activated factor VII (rFVIIa), and its use forthe treatment of blood disorders. The invention also relates to a methodfor determining whether a subject is a candidate for treatment withrFVIIa or alternative therapies. Patients respond differently to rFVIIa.The methods described herein provide a means for selecting patients withan increased likelihood of responding to rFVIIa treatment, as well aspatients for which alternative therapies may be more beneficial.

In one aspect, the invention provides a method of treating a bleedingdisorder in a subject in need thereof. The method comprisesadministering a therapeutically effective amount of rFVIIa to thesubject, wherein (a) a sample of platelets derived from the subject wasobtained; (b) a platelet population from the sample was incubated withrFVIIa; and (c) a subpopulation of platelets having at least a 4-foldhigher binding capacity to rFVIIa, as compared to the binding capacityto rFVIIa of the other platelets in the platelet population, wasdetected in the platelet population. Alternatively, the method comprisesadministering a therapeutically effective amount of rFVIIa to thesubject, wherein (a) a sample of platelets derived from the subject wasobtained; (b) a platelet population from the sample was incubated withrFVIIa; and (c) platelets having a high binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of a control plateletpopulation, were detected in the platelet population.

Also included is recombinant FVIIa for use in treating (or for use inthe preparation of a medicament for treating) a bleeding disorder in asubject in need thereof having a subpopulation of platelets (i) havingat least a 4-fold higher binding capacity to rFVIIa as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation and/or (ii) having a higher binding capacity to rFVIIa ascompared to the binding capacity to rFVIIa of a control plateletpopulation. Indeed, the invention include a method for treating ableeding disorder comprising administering rFVIIa to a subject in needthereof and having a subpopulation of platelets (i) having at least a4-fold higher binding capacity to rFVIIa as compared to the bindingcapacity to rFVIIa of the other platelets in the platelet populationand/or (ii) having a higher binding capacity to rFVIIa as compared tothe binding capacity to rFVIIa of a control platelet population.

In another aspect, the invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an alternativetherapy to the subject, wherein (a) a sample of platelets derived fromthe subject was obtained; (b) a platelet population from the sample wasincubated with rFVIIa; and (c) a subpopulation of platelets having atleast a 4-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation, was not detected in the platelet population. Alternatively,the method comprises administering a therapeutically effective amount ofan alternative therapy to the subject, wherein (a) a sample of plateletsderived from the subject was obtained; (b) a platelet population fromthe sample was incubated with rFVIIa; and (c) platelets having a highbinding capacity to rFVIIa, as compared to the binding capacity torFVIIa of a control platelet population, were not detected in theplatelet population.

Also included is an alternative therapy for use in treating (or for usein the preparation of a medicament for treating) a bleeding disorder ina subject in need thereof lacking a subpopulation of platelets (i)having at least a 4-fold higher binding capacity to rFVIIa as comparedto the binding capacity to rFVIIa of the other platelets in the plateletpopulation and/or (ii) having a higher binding capacity to rFVIIa ascompared to the binding capacity to rFVIIa of a control plateletpopulation. Indeed, the invention includes a method for treating ableeding disorder comprising administering an alternative therapy to asubject in need thereof and lacking a subpopulation of platelets (i)having at least a 4-fold higher binding capacity to rFVIIa as comparedto the binding capacity to rFVIIa of the other platelets in the plateletpopulation and/or (ii) having a higher binding capacity to rFVIIa ascompared to the binding capacity to rFVIIa of a control plateletpopulation.

In yet another aspect, the invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of aplatelet population containing (e.g., enriched with) a subpopulation ofplatelets having at least a 4-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population. Alternatively, the method comprises administeringto the subject a therapeutically effective amount of a plateletpopulation comprising platelets having a higher binding capacity torFVIIa, as compared to the binding capacity to rFVIIa of platelets in acontrol platelet population. For example, in various embodiments, theplatelets in the platelet population have at least a 0.25 fold,0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5 fold, 3-fold, 3.5-fold, 4-fold,4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold,8.5-fold, 9-fold, or higher binding capacity to rFVIIa, as compared tothe binding capacity to rFVIIa of platelets in a control plateletpopulation (or other platelets in the platelet population). Theplatelets in the control platelet population are platelets not exposedto rFVIIa.

Thus, in various aspects, a sample of platelets derived from the subjectis obtained and characterized for rFVIIa binding. In one embodiment, thesample of platelets is obtained from a blood sample from the subject. Inanother embodiment, the sample of platelets is obtained from a serumsample from the subject. The sample of platelets is, in variousembodiments, a fresh sample, a concentrate, a preserved sample, arehydrated lyophilized sample, or a frozen sample. In methods comprisingadministering a platelet population or subpopulation to a subject, theplatelet population or subpopulation is from the subject, althoughplatelets administered to a subject in the context of the invention alsomay originate from one or more other donors.

In various embodiments, a platelet population from the sample derivedfrom the subject is incubated with rFVIIa. The concentration of rFVIIais, e.g., about 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 200 nM,300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900nM, 2000 nM, 3000 nM, 4000 nM, 5000 nM, 6000 nM, or more. The plateletpopulation may be incubated with rFVIIa for any suitable amount of time,e.g., at least or no more than 7, 10, 12, 15, 20, or 30 minutes.

Detection of platelets with desired characteristics is achieved by anymethod known to one of skill in the art, such as a method that detectsrFVIIa-bound platelets. Detection is, in various embodiments, performedby flow cytometry, immunofluorescence, microscopy (such as, but notlimited to, electron microscopy or confocal laser scanning microscopy(CLSM or LSCM)), or immunoassay (such as, but not limited to, cell-basedenzyme-linked immunosorbent assay). The rFVIIa binding capacity ofplatelets is optionally measured through, e.g., detection offluorescence intensity. In one embodiment, the fluorescence intensity isproportional to the amount of rFVIIa bound to the platelets.

In one embodiment, the platelets in the platelet population (orsubpopulation of platelets, or control platelet population) areactivated. The activated platelets may be coated or not coated. Inanother embodiment, the platelets in the platelet population (orsubpopulation of platelets, or control platelet population) arenon-activated. In one embodiment, the platelets in the plateletpopulation (or subpopulation of platelets, or control plateletpopulation) are coated. In another embodiment, the platelets in theplatelet population (or subpopulation of platelets, or control plateletpopulation) are not coated.

In one embodiment, the platelets in the platelet population or controlplatelet population have a rFVIIa binding constant of about 25 to 1100nM. In another embodiment, the platelets in the platelet population orcontrol platelet population have a rFVIIa binding constant of about 50to 400 nM. In further embodiments, the platelets in the plateletpopulation or control platelet population have a rFVIIa binding constantof about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM,about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about700 nM, about 800 nM, about 900 nM, about 1000 nM, about 1100 nM, about1200 nM, about 1300 nM, about 1400 nM, about 1500 nM, about 1600 nM,about 1700 nM, about 1800 nM, about 1900 nM, about 2000 nM, about 3000nM, about 4000 nM, or about 5000 nM, about 6000 nM, about 7000 nM, about8000 nM, about 9000 nM, about 10,000 nM, or more.

In other embodiments, the platelets in the platelet population orcontrol platelet population have a rFVIIa binding constant of about 10nM to 30 nM, about 20 nM to 40 nM, about 30 nM to 50 nM, about 40 nM to60 nM, about 50 nM to 70 nM, about 60 nM to 80 nM, about 70 nM to 90 nM,about 80 nM to 100 nM, 90 nM to 110 nM, about 100 nM to 120 nM, about110 nM to 130 nM, about 120 nM to 140 nM, about 130 nM to 150 nM, about140 nM to 160 nM, about 150 nM to 170 nM, about 160 nM to 180 nM, about170 nM to 190 nM, about 180 nM to 200 nM, about 190 nM to 210 nM, about200 nM to 220 nM, about 210 nM to 230 nM, about 220 nM to 240 nM, about230 nM to 250 nM, about 240 nM to 260 nM, about 250 nM to 270 nM, about260 nM to 280 nM, about 270 nM to 290 nM, about 280 nM to 300 nM, about290 nM to 310 nM, about 300 nM to 320 nM, about 310 nM to 330 nM, about320 nM to 340 nM, about 330 nM to 350 nM, about 340 nM to 360 nM, about350 nM to 370 nM, about 360 nM to 380 nM, about 370 nM to 390 nM, about380 nM to 400 nM, about 390 nM to 410 nM, about 400 nM to 450 nM, about450 nM to 500 nM, about 500 nM to 550 nM, about 550 nM to 600 nM, about600 nM to 650 nM, about 650 nM to 700 nM, about 700 nM to 750 nM, about750 nM to 800 nM, about 800 nM to 850 nM, about 850 nM to 900 nM, about900 nM to 950 nM, about 950 nM to 1000 nM, about 1000 nM to 1050 nM,about 1050 nM to 1100 nM, about 1100 nM to 1150 nM, about 1150 nM to1200 nM, about 1200 nM to 1250 nM, about 1250 nM to 1300 nM, about 1300nM to 1350 nM, about 1350 nM to 1400 nM, about 1400 nM to 1450 nM, orabout 1450 nM to 1500 nM, or any range in between.

In one embodiment, the platelets in the subpopulation of platelets orthe platelets having a higher rFVIIa binding capacity than a controlpopulation have a rFVIIa binding constant of about 10 nM, about 20 nM,about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900nM, about 1000 nM, about 1100 nM, about 1200 nM, about 1300 nM, about1400 nM, about 1500 nM, about 1600 nM, about 1700 nM, about 1800 nM,about 1900 nM, about 2000 nM, about 3000 nM, about 4000 nM, about 5000nM, about 6000 nM, about 7000 nM, about 8000 nM, about 9000 nM, about10,000 nM, or more.

In various embodiments, the platelets in the subpopulation of plateletsor the platelets having a higher rFVIIa binding capacity than a controlpopulation have a rFVIIa binding constant of about 10 nM to 30 nM, about20 nM to 40 nM, about 30 nM to 50 nM, about 40 nM to 60 nM, about 50 nMto 70 nM, about 60 nM to 80 nM, about 70 nM to 90 nM, about 80 nM to 100nM, 90 nM to 110 nM, about 100 nM to 120 nM, about 110 nM to 130 nM,about 120 nM to 140 nM, about 130 nM to 150 nM, about 140 nM to 160 nM,about 150 nM to 170 nM, about 160 nM to 180 nM, about 170 nM to 190 nM,about 180 nM to 200 nM, about 190 nM to 210 nM, about 200 nM to 220 nM,about 210 nM to 230 nM, about 220 nM to 240 nM, about 230 nM to 250 nM,about 240 nM to 260 nM, about 250 nM to 270 nM, about 260 nM to 280 nM,about 270 nM to 290 nM, about 280 nM to 300 nM, about 290 nM to 310 nM,about 300 nM to 320 nM, about 310 nM to 330 nM, about 320 nM to 340 nM,about 330 nM to 350 nM, about 340 nM to 360 nM, about 350 nM to 370 nM,about 360 nM to 380 nM, about 370 nM to 390 nM, about 380 nM to 400 nM,about 390 nM to 410 nM, about 400 nM to 450 nM, about 450 nM to 500 nM,about 500 nM to 550 nM, about 550 nM to 600 nM, about 600 nM to 650 nM,about 650 nM to 700 nM, about 700 nM to 750 nM, about 750 nM to 800 nM,about 800 nM to 850 nM, about 850 nM to 900 nM, about 900 nM to 950 nM,about 950 nM to 1000 nM, about 1000 nM to 1050 nM, about 1050 nM to 1100nM, about 1100 nM to 1150 nM, about 1150 nM to 1200 nM, about 1200 nM to1250 nM, about 1250 nM to 1300 nM, about 1300 nM to 1350 nM, about 1350nM to 1400 nM, about 1400 nM to 1450 nM, or about 1450 nM to 1500 nM, orany range in between.

Recombinant FVIIa binding capacity is compared to other platelets in theplatelet population and/or platelets in a control platelet population(platelets that have not been exposed to rFVIIa). In an exemplaryembodiment, binding capacity is determined by, e.g., measuringfluorescence intensity (optionally via flow cytometry), which isproportional to the amount of rFVIIa bound to the platelets. Plateletsare identified for flow cytometry by their forward and side scatterlight properties, and by expression of CD61, a platelet-specific surfacemarker protein. Activated platelets are indentified by using increase ofsurface-bound CD62P (P-selectin), and coated platelets are identified byusing fibrinogen as surface marker. These surface-bound proteins can beidentified using fluorochrome-conjugated antibodies specific for therespective protein. By using multiparameter flow cytometry, theplatelets binding rFVIIa and, for example, exposing CD62P and fibrinogencan be detected. Platelet populations and subpopulations can beidentified using signal intensity of any relevant channel by makingso-called dot-plots for data anylsis. Additionally, platelets can beplotted in a histogram showing the distribution of the platelets interms of amount of rFVIIa bound to the surface. A subpopulation can beidentified if two separate peaks can be observed, or if a “shoulder” isvisible. Especially in case of a “shoulder” in the histogram, a dot-plotcan be made to add another criterion for discrimination, e.g.side-scatter or CD62P. A subpopulation demonstrating a higher medianfluorescence intensity than the median fluorescence intensity of theremainder of the platelet population (or a control platelet population),and which optionally also represents at least about 2% of the totalplatelet population examined, is a subpopulation of platelets having ahigher rFVIIa binding capacity (i.e., binds more rFVIIa) compared to themajor population (or a control population).

In one embodiment, the platelet population contains a subpopulation ofplatelets having about a 0.1-fold, about a 0.15-fold, about a 0.2-fold,about a 0.25-fold, about a 0.3-fold, about a 0.4-fold, about a 0.5 fold,about a 0.6-fold, about a 0.7-fold, about a 0.8-fold, about a 0.9-fold,about a 1-fold, about a 1.5-fold, about a 2 fold, about a 2.5-fold,about a 3-fold, about a 3.5-fold, about a 4-fold, about a 4.5-fold,about a 5-fold, about a 5.5-fold, about a 6-fold, about a 6.5-fold,about a 7-fold, about a 7.5-fold, about a 8-fold, about a 8.5-fold, orabout a 9-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation. In another embodiment, the platelet population contains asubpopulation of platelets having at least about a 10-fold, about a11-fold, about a 12 fold, about a 13-fold, about a 14-fold, about a15-fold, about a 16-fold, about a 17-fold, about a 18-fold, about a19-fold, about a 20-fold, about a 21-fold, about a 22-fold, about a23-fold, about a 24-fold, about a 25-fold, about a 26-fold, about a27-fold, about a 28-fold, about a 29-fold, about a 30-fold, about a31-fold, about a 32-fold, about a 33-fold, about a 34-fold, about a35-fold, about a 36-fold, about a 37-fold, about a 38-fold, about a39-fold, about a 40-fold, about a 41-fold, about a 42-fold, about a43-fold, about a 44-fold, about a 45-fold, about a 46-fold, about a47-fold, about a 48-fold, about a 49-fold, about a 50-fold, about a51-fold, about a 52-fold, about a 53-fold, about a 54-fold, about a55-fold, about a 56-fold, about a 57-fold, about a 58-fold, about a59-fold, about a 60-fold, about a 70-fold, about a 80-fold, about a90-fold, about a 100-fold, about a 150-fold, about a 200-fold, about a250-fold, about a 300-fold, about a 350-fold, about a 400-fold, about a450-fold, or about a 500-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population.

In another embodiment, the platelets in the subpopulation have about a0.3-fold to 2.5-fold higher binding capacity (e.g., about a 0.3-fold to2.4-fold higher binding capacity), about a 4-fold to 33-fold higherbinding capacity, or about a 5-fold to 37-fold higher binding capacity,as compared to the binding capacity to rFVIIa of platelets in the restof the population.

In another embodiment, the platelet population contains a subpopulationof platelets having about a 0.1-fold to 0.3-fold, about a 0.2-fold to0.4-fold, about a 0.3-fold to 0.5-fold, about a 0.4-fold to 0.6-fold,about a 0.5-fold to 0.7-fold, about a 0.6-fold to 0.8-fold, about a0.7-fold to 0.9-fold, about a 0.8-fold to 1.0-fold, about a 0.9-fold to1.1-fold, about a 1-fold to 1.5-fold, about a 1.25-fold to 1.75-fold,about a 1.5-fold to 2-fold, about a 1.75-fold to 2.25-fold, about a2-fold to 2.5-fold, about a 2.25-fold to 2.75-fold, about a 2.5-fold to3-fold, about a 2.75-fold to 3.25-fold, about a 3-fold to 4-fold, abouta 3.5-fold to 4.5-fold, about a 4-fold to 5-fold, about a 4.5-fold to5.5-fold, about a 5-fold to 6-fold, about a 6-fold to 8-fold, about a8-fold to 10-fold, about a 10-fold to 12-fold, about a 12-fold to14-fold, about a 14-fold to 16-fold, about a 16-fold to 18-fold, about a18-fold to 20-fold, about a 20-fold to 25-fold, about a 25-fold to30-fold, about a 30-fold to 40-fold, about a 40-fold to 50-fold, about a50-fold to 70-fold, about a 70-fold to 90-fold, about a 90-fold to100-fold, about a 100-fold to 120-fold, about a 120-fold to 140-fold,about a 140-fold to 160-fold, about a 1-fold to 20-fold, about a 10-foldto 30-fold, about a 20-fold to 40-fold, about a 30-fold to 50-fold,about a 40-fold to 60-fold, about a 50-fold to 80-fold, about a 60-foldto 80-fold, about a 70-fold to 90-fold, about a 80-fold to 100-fold,about a 100-fold to 150-fold, about a 130-fold to 180-fold, or any rangein between, higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation.

In one embodiment, the platelets having a high binding capacity torFVIIa compared to a control population have about a 0.1-fold, about a0.15-fold, about a 0.2-fold, about a 0.25-fold, about a 0.3-fold, abouta 0.4-fold, about a 0.5-fold, about a 0.6-fold, about a 0.7-fold, abouta 0.8-fold, about a 0.9-fold, about a 1-fold, about a 1.5-fold, about a2 fold, about a 2.5-fold, about a 3-fold, about a 3.5-fold, about a4-fold, about a 4.5-fold, about a 5-fold, about a 5.5-fold, about a6-fold, about a 6.5-fold, about a 7-fold, about a 7.5-fold, about a8-fold, about a 8.5-fold, or about a 9-fold higher binding capacity torFVIIa, as compared to the binding capacity to rFVIIa of platelets in acontrol platelet population. In another embodiment, the platelets havinga high binding capacity to rFVIIa have at least about a 10-fold, about a11-fold, about a 12-fold, about a 13-fold, about a 14-fold, about a15-fold, about a 16-fold, about a 17-fold, about a 18-fold, about a19-fold, about a 20-fold, about a 21-fold, about a 22-fold, about a23-fold, about a 24-fold, about a 25-fold, about a 26-fold, about a27-fold, about a 28-fold, about a 29-fold, about a 30-fold, about a31-fold, about a 32-fold, about a 33-fold, about a 34-fold, about a35-fold, about a 36-fold, about a 37-fold, about a 38-fold, about a39-fold, about a 40-fold, about a 41-fold, about a 42-fold, about a43-fold, about a 44-fold, about a 45-fold, about a 46-fold, about a47-fold, about a 48-fold, about a 49-fold, about a 50-fold, about a51-fold, about a 52-fold, about a 53-fold, about a 54-fold, about a55-fold, about a 56-fold, about a 57-fold, about a 58-fold, about a59-fold, about a 60-fold, about a 70-fold, about a 80-fold, about a90-fold, about a 100-fold, about a 150-fold, about a 200-fold, about a250-fold, about a 300-fold, about a 350-fold, about a 400-fold, about a450-fold, or about a 500-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of platelets in a controlplatelet population.

In another embodiment, the platelets in the platelet population having ahigh rFVIIa binding capacity have about a 0.3-fold to 2.5-fold higherbinding capacity (e.g., about a 0.3-fold to 2.4-fold higher bindingcapacity), about a 4-fold to 33-fold higher binding capacity, or about a5-fold to 37-fold higher binding capacity, as compared to the bindingcapacity to rFVIIa of platelets in a control platelet population.

In another embodiment, the platelets in the platelet population having ahigh binding capacity to rFVIIa have about a 0.1-fold to 0.3-fold, abouta 0.2-fold to 0.4-fold, about a 0.3-fold to 0.5-fold, about a 0.4-foldto 0.6-fold, about a 0.5-fold to 0.7-fold, about a 0.6-fold to 0.8-fold,about a 0.7-fold to 0.9-fold, about a 0.8-fold to 1.0-fold, about a0.9-fold to 1.1-fold, about a 1-fold to 1.5-fold, about a 1.25-fold to1.75-fold, about a 1.5-fold to 2-fold, about a 1.75-fold to 2.25-fold,about a 2-fold to 2.5-fold, about a 2.25-fold to 2.75-fold, about a2.5-fold to 3-fold, about a 2.75-fold to 3.25-fold, about a 3-fold to4-fold, about a 3.5-fold to 4.5-fold, about a 4-fold to 5-fold, about a4.5-fold to 5.5-fold, about a 5-fold to 6-fold, about a 6-fold to8-fold, about a 8-fold to 10-fold, about a 10-fold to 12-fold, about a12-fold to 14-fold, about a 14-fold to 16-fold, about a 16-fold to18-fold, about a 18-fold to 20-fold, about a 20-fold to 25-fold, about a25-fold to 30-fold, about a 30-fold to 40-fold, about a 40-fold to50-fold, about a 50-fold to 70-fold, about a 70-fold to 90-fold, about a90-fold to 100-fold, about a 100-fold to 120-fold, about a 120-fold to140-fold, about a 140-fold to 160-fold, about a 1-fold to 20-fold, abouta 10-fold to 30-fold, about a 20-fold to 40-fold, about a 30-fold to50-fold, about a 40-fold to 60-fold, about a 50-fold to 80-fold, about a60-fold to 80-fold, about a 70-fold to 90-fold, about a 80-fold to100-fold, about a 100-fold to 150-fold, about a 130-fold to 180-fold, orany range in between higher binding capacity to rFVIIa, as compared tothe binding capacity to rFVIIa of platelets in a control plateletpopulation. The “control” platelet population is a population notexposed to rFVIIa prior to examining binding characteristics.

In various embodiments, the platelet population contains a subpopulationof platelets having about a 50%, 75%, 100%, 125%, 150%, 175%, 200%,300%, 400%, or a 500% higher binding capacity to rFVIIa, as compared tothe binding capacity to rFVIIa of the other platelets in the plateletpopulation. The platelets having a higher binding capacity to rFVIIacompared to platelets in a control population have about a 50%, 75%,100%, 125%, 150%, 175%, 200%, 300%, 400%, or a 500% higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of thecontrol platelet population.

In one embodiment, the platelet subpopulation with a higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of theother platelets in the platelet population, consists of at least 2% ofthe platelet population. In various embodiments, the plateletsubpopulation represents (consists of) at least 0.5%, 1%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, or more(e.g., 60%, 70%, 75%, 80%, 90%, 95%, or 100%) of the plateletpopulation.

The invention also includes the subpopulation of platelets describedherein. For example, the invention includes a subpopulation of plateletsisolated from a platelet population, wherein the platelets in thesubpopulation of platelets have at least a 4-fold higher bindingcapacity to rFVIIa as compared to the binding capacity to rFVIIa of theother platelets in the platelet population. Detection of thesubpopulation and characterization of rFVIIa binding capacity isdescribed above and applicable here. The platelets in the subpopulationof platelets are activated or non-activated. The platelets in thesubpopulation also may be coated or not coated.

In various embodiments, the bleeding disorder is hemophilia (e.g.,hemophilia with inhibitors or hemophilia without inhibitors). In variousembodiments, the hemophilia is hemophilia A (e.g., hemophilia A withinhibitors) or hemophilia B (e.g., hemophilia B with inhibitors). Theinhibitors are optionally to Factor VIII or Factor IX. In anotherembodiment, the hemophilia is acquired hemophilia. In one embodiment,the hemophilia is congenital hemophilia A with inhibitors or acquiredhemophilia A with inhibitory auto antibodies to FVIII. In oneembodiment, the hemophilia is congenital hemophilia B with inhibitors oracquired hemophilia B with inhibitory auto antibodies to FIX. Inaddition, the rFVIIa is optionally administered if the subject does nothave an unacceptable risk of thrombosis.

In alternative embodiments, the bleeding disorder is a non-hemophiliableeding disorder. In one embodiment, the bleeding disorder is bloodloss from trauma or blood loss from surgery (e.g., high risk surgery).Other examples of bleeding disorders include, but are not limited to,FVII deficiency, FV deficiency, FX deficiency, FXI deficiency, FXIIIdeficiency, fibrinogen deficiency, prothrombin deficiency, dilutionalcoagulopathy, thrombocytopenia, and von Willebrand disease (e.g., vonWillebrand disease with inhibitors to von Willebrand factor). In anotherembodiment, the bleeding disorder is intracerebral hemorrhage.

In one embodiment, the bleeding disorder is a congenital plateletfunction defect, including, but not limited to, platelet storage pooldisorder, Glanzmann's thrombasthenia, or Bernard-Soulier syndrome. Inone embodiment, the bleeding disorder is an acquired platelet functiondefect. In one embodiment, the bleeding disorder is a congenitaldeficiency of Factor II, Factor V, Factor VII, Factor X, or Factor XI.In one embodiment, the bleeding disorder is a neonatal or pediatriccoagulopathy. In one embodiment, the bleeding disorder is a plateletfunction disorder. In another embodiment, the bleeding disorder isheparin-induced thrombocytopenia. In one embodiment, the bleedingdisorder is disseminated intravascular coagulation. In otherembodiments, the bleeding disorder is any disorder known to one of skillin the art (for additional information, see e.g., The Absite Review, bySteven M. Fiser, Lippincott Williams and Wilkins 2004).

In various aspects, the inventive method comprises administering to asubject a therapeutically effective amount of recombinant Factor VIla(rFVIIa). Examples of suitable rFVIIa include, but are not limited to,NovoSeven® (Novo Nordisk) and BAX817. The rFVIIa is optionallyadministered with, or part of a therapeutic regimen that also includes,FX, ATIII (antithrombin III), a combination of FX and ATIII, FII, FX, acombination of FII and FX, and platelet infusion (optionally infusion ofplatelets described herein).

In various aspects, the method comprises administering an alternativetherapy, i.e., therapy other than rFVIIa. In one embodiment, thealternative therapy is a therapy that is used for treating a bleedingdisorder and that is not rFVIIa (e.g., not rFVIIa as a single agent) ordoes not comprise rFVIIa, as determined by a person of skill in the art.For additional information on therapies to treat bleeding disorders,including dosage and administration, see e.g., The Absite Review, bySteven M. Fiser, Lippincott Williams and Wilkins 2004.

In one embodiment, the alternative therapy is a coagulation factor thatis not recombinant rFVIIa, or a variant of a coagulation factor that isnot recombinant rFVIIa. The coagulation factor is recombinant ornon-recombinant, and is activated or non-activated. Exemplary non-FVIIacoagulation factors include, but are not limited, to FI (fibrinogen),FII (prothrombin), FIII (tissue factor), FIV, FV, FVa, FVIII, FIX, FX,FXa, FXI, FXIII, von Willebrand factor, prekallikrein, andhigh-molecular weight kininogen. In various embodiments, the alternativetherapy is a mixture of coagulation factors, e.g., two, three, or morecoagulation factors, one or more of which is a recombinant, activated,or non-activated coagulation factors.

In one embodiment, the alternative therapy is BeneFix® (recombinantFactor IX), Rixubis® (recombinant Factor IX) Kogenate® FS (recombinantFactor VIII), Recombinate (recombinant Factor VIII), Advate®(recombinant Factor VIII), Helixate® FS (recombinant Factor VIII),Koāte®-DVI (recombinant Factor VIII), Stimate® (desmopressin acetate),DDAVP® (desmopressin acetate), Bebulin (Factor IX Complex),cryoprecipitated antihaemophilic factor (AHF), Octanate® (human FactorVIII/von Willebrand Factor (VWF)), Hemofil M® (human factor VIII), orfresh frozen plasma (FFP) (or any combination of two or more of theforegoing). For additional information on therapies to treat bleedingdisorders, including dosage and administration, see e.g., The AbsiteReview, by Steven M. Fiser, Lippincott Williams and Wilkins 2004.

The term “Benefix®” is a brand name for the drug Coagulation Factor IX(Recombinant). Indications, dosage and methods of administration of thisdrug are known to one of skill in the art. For additional information,see www.benefix.com.

“Rixubis®” is a brand name for a recombinant Factor IX product. Foradditional information, see www.rixubis.com.

The term “Kogenate® FS” is a brand name for a recombinant factor VIIIproduct. Indications, dosage and methods of administration of this drugare known to one of skill in the art. For additional information, seewww.kogenate.com.

The term “Recombinate” is a brand name for a drug that is a recombinantantihemophilic factor. Indications, dosage and methods of administrationof this drug are known to one of skill in the art. For additionalinformation, see www.recombinate.com.

The term “Advate®” designates a drug that is a recombinantantihemophilic factor, used to replace clotting factor VIII.Indications, dosage and methods of administration of this drug are knownto one of skill in the art. For additional information, seewww.advate.com.

The term “Helixate® FS” is a brand name for a drug that is a recombinantfactor VIII treatment. Indications, dosage and methods of administrationof this drug are known to one of skill in the art. For additionalinformation, see www.helixatefs.com.

The term “Koāte®-DVI” is a brand name for a drug that is a humanantihemophilic factor treatment. Indications, dosage and methods ofadministration of this drug are known to one of skill in the art. Foradditional information, see www.koate-dvi.com/.

The term “Stimate®” is a brand of desmopressin used to help stopbleeding in patients with von Willebrand's disease or mild hemophilia A.Indications, dosage and methods of administration of this drug are knownto one of skill in the art. For additional information, seewww.stimate.com/.

The term “DDAVP®” is a brand of desmopressin used to help stop bleedingin patients with von Willebrand's disease or mild hemophilia A.Indications, dosage and methods of administration of this drug are knownto one of skill in the art.

The term “Bebulin” designates the drug Factor IX Complex. Indications,dosage and methods of administration of this drug are known to one ofskill in the art. For additional information, see www.baxter.com.

The term “Hemofil M®” is a brand of antihemophilic human factor VIII.Indications, dosage and methods of administration of this drug are knownto one of skill in the art. For additional information, seewww.baxter.com.

In one embodiment, the alternative therapy is recombinant porcine FVIIIa recombinant FV variant, a recombinant FVIIa variant, a recombinant FXavariant, FXIII, prothrombin, or fibrinogen. In another embodiment, thealternative therapy is a mix of coagulation factors, e.g., a mix of FX(plasma-derived or recombinant FX), FVIIa (plasma-derived or recombinantFVIIa), and antithrombin III (ATIII). Other suitable alternativetherapies include, but are not limited to, antibodies mimicking FVIII,peptides mimicking FVIII, and compounds mimicking FVIII. In oneembodiment, the alternative therapy is a peptide inhibitor of TFPI, anantibody inhibitor of TFPI, or a compound inhibiting TFPI. In anotherembodiment, the alternative therapy is compounds inhibitinganti-coagulant proteins or agents that reduce expression ofanti-coagulant proteins.

In one embodiment, the alternative therapy is a molecule that can mimica coagulation factor, a molecule that can mimic the activity of acoagulation factor, or a molecule which has procoagulation activity. Ina further embodiment, the alternative therapy can be a mixture ofmolecules that mimic coagulation factors, or the activity of coagulationfactors. In one embodiment, the molecule is a small molecule, a peptide,or an antibody or a fragment thereof.

In one embodiment, the alternative therapy is an inhibitor of ananti-coagulant. In one embodiment, the inhibitor can include, but is notlimited to, a small molecule, a peptide, or an antibody. In oneembodiment the anti-coagulant can include, but is not limited to proteinC, heparin cofactor II, heparin cofactor III, anti-thrombin, protein Z,protein S, protein Z-related protease inhibitor, plasminogen, alpha2-antiplasmin, tissue plasminogen activator, urokinase, plasminogenactivator inhibitor-1, plasminogen activator inhibitor-2, or cancerprocoagulant. In a further embodiment, the alternative therapy can be amixture of inhibitors of an anti-coagulant.

In one embodiment, the alternative therapy is Prothrombin ComplexConcentrate or activated Prothrombin Complex Concentrate. In oneembodiment, the activated Prothrombin Complex Concentrate is FEIBA. Theterm “FEIBA” designates the drug Factor VIII Inhibitor BypassingComplex, or Anti-Inhibitor Coagulant Complex. Indications, dosage andmethods of administration of this drug are known to one of skill in theart. For additional information, see www.feiba.com.

In one embodiment, the subject is an animal, such as a mammal. Invarious embodiments, the subject is a human. In some embodiments, thesubject is a rodent, such as a mouse or a rat. In some embodiments, thesubject is a cow, pig, sheep, goat, cat, horse, dog, and/or any otherspecies of animal used as livestock or kept as pets.

Optionally, the subject is suspected to have a bleeding disorder, hasbeen diagnosed with a bleeding disorder, is predisposed to developing ableeding disorder, or is at risk of developing a bleeding disorder. Inone embodiment, the subject is being treated for a bleeding disorderbefore initiation of the inventive method. In other embodiments, thesubject has not previously been treated for a bleeding disorder prior tothe inventive method.

The subject has a risk of thrombosis or does not have a risk ofthrombosis. In one embodiment, the subject has an unacceptable risk ofthrombosis. In another embodiment, the subject does not have anunacceptable risk of thrombosis (although this is not required). Forexample, in various embodiments, the bleeding disorder is anon-hemophilia bleeding disorder, and rFVIIa is administered if thesubject does not have an unacceptable risk of thrombosis.

The invention provides a method of treating a bleeding disorder in asubject in need thereof, the method comprising administering atherapeutically effective amount of rFVIIa to the subject, wherein (a) asample of platelets derived from the subject was obtained; (b) aplatelet population from the sample was incubated with rFVIIa; and (c)platelets having a high binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of a control platelet population, weredetected in the platelet population. The invention also provides amethod of treating a bleeding disorder in a subject in need thereof, themethod comprising administering a therapeutically effective amount ofrFVIIa to the subject, wherein (a) a sample of platelets derived fromthe subject was obtained; (b) a platelet population from the sample wasincubated with rFVIIa; and (c) platelets having a low binding capacityto rFVIIa, as compared to the binding capacity to rFVIIa of a controlplatelet population, were detected in the platelet population.

In another aspect, the invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an alternativetherapy to the subject, wherein (a) a sample of platelets derived fromthe subject was obtained; (b) a platelet population from the sample wasincubated with rFVIIa; and (c) platelets having a high binding capacityto rFVIIa, as compared to the binding capacity to rFVIIa of a controlplatelet population, were detected in the platelet population. Theinvention also provides a method of treating a bleeding disorder in asubject in need thereof, the method comprising administering atherapeutically effective amount of an alternative therapy to thesubject, wherein (a) a sample of platelets derived from the subject wasobtained; (b) a platelet population from the sample was incubated withrFVIIa; and (c) platelets having a low binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of a control plateletpopulation, were detected in the platelet population.

In various embodiments, the platelets having a high binding capacity torFVIIa have about a 25-fold higher or about a 35-fold higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of acontrol platelet population. In various embodiments, the plateletshaving a low binding capacity to rFVIIa have about a 5-fold higher orabout a 10-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of a control platelet population.

In one embodiment, the platelets having a low binding capacity to rFVIIahave about a 0.1-fold, about a 0.15-fold, about a 0.2-fold, about a0.25-fold, about a 0.3-fold, about a 0.4-fold, about a 0.5 fold, about a0.6-fold, about a 0.7-fold, about a 0.8-fold, about a 0.9-fold, about a1-fold, about a 1.5-fold, about a 2 fold, about a 2.5-fold, about a3-fold, about a 3.5-fold, about a 4-fold, about a 4.5-fold, about a5-fold, about a 5.5-fold, about a 6-fold, about a 6.5-fold, about a7-fold, about a 7.5-fold, about a 8-fold, about a 8.5-fold, or about a9-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of a control platelet population. In anotherembodiment, the platelets having a low binding capacity to rFVIIa haveabout a 10-fold, about a 11-fold, about a 12 fold, about a 13-fold,about a 14-fold, about a 15-fold, about a 16-fold, about a 17-fold,about a 18-fold, about a 19-fold, about a20-fold, about a 21-fold, abouta 22-fold, about a 23-fold, about a 24-fold, about a 25-fold, about a26-fold, about a 27-fold, about a 28-fold, about a 29-fold, about a30-fold, about a 31-fold, about a 32-fold, about a 33-fold, about a34-fold, about a 35-fold, about a 36-fold, about a 37-fold, about a38-fold, about a 39-fold, about a 40-fold, about a 41-fold, about a42-fold, about a 43-fold, about a 44-fold, about a 45-fold, about a46-fold, about a 47-fold, about a 48-fold, about a 49-fold, about a50-fold, about a 51-fold, about a 52-fold, about a 53-fold, about a54-fold, about a 55-fold, about a 56-fold, about a 57-fold, about a58-fold, about a 59-fold, about a 60-fold, about a 70-fold, about a80-fold, about a 90-fold, about a 100-fold, about a 150-fold, about a200-fold, about a 250-fold, about a 300-fold, about a 350-fold, about a400-fold, about a 450-fold, or about a 500-fold higher binding capacityto rFVIIa, as compared to the binding capacity to rFVIIa of a controlplatelet population.

In another embodiment, the platelets having a low binding capacity torFVIIa have about a 0.1-fold to 0.3-fold, about a 0.2-fold to 0.4-fold,about a 0.3-fold to 0.5-fold, about a 0.4-fold to 0.6-fold, about a0.5-fold to 0.7-fold, about a 0.6-fold to 0.8-fold, about a 0.7-fold to0.9-fold, about a 0.8-fold to 1.0-fold, about a 0.9-fold to 1.1-fold,about a 1-fold to 1.5-fold, about a 1.25-fold to 1.75-fold, about a1.5-fold to 2-fold, about a 1.75-fold to 2.25-fold, about a 2-fold to2.5-fold, about a 2.25-fold to 2.75-fold, about a 2.5-fold to 3-fold,about a 2.75-fold to 3.25-fold, about a 3-fold to 4-fold, about a abouta 3.5-fold to 4.5-fold, about a 4-fold to 5-fold, about a 4.5-fold to5.5-fold, about a 5-fold to 6-fold, about a 6-fold to 8-fold, about a8-fold to 10-fold, about a 10-fold to 12-fold, about a 12-fold to14-fold, about a 14-fold to 16-fold, about a 16-fold to 18-fold, about a18-fold to 20-fold, about a 20-fold to 25-fold, about a 25-fold to30-fold, about a 30-fold to 40-fold, about a 40-fold to 50-fold, about a50-fold to 70-fold, about a 70-fold to 90-fold, about a 90-fold to100-fold, about a 100-fold to 120-fold, about a 120-fold to 140-fold,about a 140-fold to 160-fold, about a 1-fold to 20-fold, about a 10-foldto 30-fold, about a 20-fold to 40-fold, about a 30-fold to 50-fold,about a 40-fold to 60-fold, about a 50-fold to 80-fold, about a 60-foldto 80-fold, about a 70-fold to 90-fold, about a 80-fold to 100-fold,about a 100-fold to 150-fold, about a 130-fold to 180-fold, or any rangein between, higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of a control platelet population.

In another embodiment, the platelets having a low binding capacity torFVIIa have about a 50%, 75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%,or a 500% higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of a control platelet population.

The invention also provides a method of treating a bleeding disorder ina subject in need thereof. The method comprises administering to thesubject a therapeutically effective amount of a platelet population,wherein the platelets in the platelet population demonstrate at least a15-fold higher rFVIIa relative fluorescence after exposure to 100 nMrFVII. Optionally, the platelet population comprises non-activatedplatelets demonstrating at least a 25-fold higher rFVIIa relativefluorescence after exposure to 100 nM rFVIIa. Also optionally, theplatelet population comprises activated platelets demonstrating at leasta 20-fold higher rFVIIa relative fluorescence after exposure to 100 nMrFVIIa, e.g., at least a 30-fold higher rFVIIa relative fluorescence,after exposure to 100 nM rFVIIa. “rFVIIa relative fluorescence” refersto the median fluorescence increase associated with rFVIIa binding toplatelets in a sample compared to a fluorescence detected in a buffercontrol (e.g., a sample of the buffer used to prepare the plateletsample for fluorescence detection). Methods of detecting and quantifyingfluorescence associated with rFVIIa binding is described herein andunderstood in the art. Any suitable exposure time to rFVIIa (e.g., 7,10, 15, or 20 minute incubation times) is appropriate.

Further, rFVIIa relative fluorescence may be employed to select subjectswith enhanced ability to respond to rFVIIa treatment or subjects with apredisposition to low responses to rFVIIa treatment. In this regard, themethods of the invention provide valuable insight to a clinician whenpreparing therapeutic regimen, as a “high responder” (i.e., a subjecthaving platelets with increased capacity for rFVIIa binding) may requirelower doses or fewer administrations of rFVIIa, while a “low responder”(i.e., a subject having platelets with reduced ability to bind rFVIIa)may require higher doses or more administrations of treatment.Similarly, “low responders” may benefit from alternative treatment, suchas the alternative treatments described herein.

In one aspect, the invention provides a method of treating a bleedingdisorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of rFVIIa to thesubject, wherein (a) a sample of platelets derived from the subject wasobtained; (b) a non-activated platelet population from the sample wasincubated with rFVIIa; and (c) platelets demonstrating at least a15-fold higher rFVIIa relative fluorescence after exposure to 100 nMrFVIIa were detected in the platelet population. Optionally,non-activated platelets demonstrating at least a 11-fold, at least a14-fold (e.g., at least a 15.5-fold or 18.7-fold) higher, at least a20-fold higher, at least a 25-fold higher, at least a 30-fold higher(e.g., at least a 32.8-fold higher), at least a 35-fold higher, at leasta 40-fold higher, or at least a 50-fold higher rFVIIa relativefluorescence is detected. Alternatively or in addition, (a) a sample ofplatelets derived from the subject was obtained; (b) an activatedplatelet population from the sample was incubated with rFVIIa; and (c)platelets demonstrating at least a 20-fold higher rFVIIa relativefluorescence after exposure to 100 nM rFVIIa were detected in theplatelet population. Optionally, activated platelets demonstrating atleast a 13-fold higher, at least a 14-fold higher, at least a 20.5-foldhigher, at least a 25-fold higher, at least a 30-fold higher (e.g., atleast a 34.3-fold higher), at least a 35-fold higher, at least a 40-foldhigher, at least a 45-fold higher, at least a 50-fold higher, or atleast a 60-fold higher rFVIIa relative fluorescence is detected.

The invention also includes a method of treating a bleeding disorder ina subject in need thereof, the method comprising administering atherapeutically effective amount of an alternative therapy to thesubject, wherein a sample of platelets derived from the subject wasobtained; a non-activated platelet population from the sample wasincubated with rFVIIa; and platelets demonstrating an 8-fold higherrFVIIa relative fluorescence or less after exposure to 100 nM rFVIIawere detected in the platelet population. In this regard, platelets inthe sample demonstrate a no more than 8-fold increase in rFVIIa relativefluorescence (i.e., demonstrate a 7-fold, a 6.5-fold, a 6-fold, a5.5-fold, a 5-fold, a 4.5-fold, a 4-fold, a 3.5-fold, a 2-fold, a1.5-fold, or less increase). Alternatively or in addition, a sample ofplatelets derived from the subject was obtained; an activated plateletpopulation from the sample was incubated with rFVIIa; and plateletsdemonstrating a 9-fold higher rFVIIa relative fluorescence or less,after exposure to 100 nM rFVIIa were detected in the plateletpopulation. Optionally, activated or non-activated platelets demonstratea 8-fold higher rFVIIa relative fluorescence or less, a 6-fold higherrFVIIa relative fluorescence or less, a 5-fold higher rFVIIa relativefluorescence or less, a 4-fold higher rFVIIa relative fluorescence orless, a 3-fold higher rFVIIa relative fluorescence or less, or a 2-foldhigher rFVIIa relative fluorescence or less. Any of the ranges describedabove with respect to higher or lower rFVIIa binding capacity also applyto rFVIIa relative fluorescence.

The increase in rFVIIa binding upon platelet activation (dual-agonistactivation) also is a suitable for selecting subjects for a particulartherapeutic regimen. The invention provides a method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of rFVIIa to a subjecthaving platelets that demonstrate at least a 2.0-fold increase, at leasta 3.0-fold increase, or at least a 3.5-fold increase in binding capacityupon dual-agonist activation compared to rFVIIa binding capacity ofnon-activated platelets from the subject. Optionally, the plateletsdemonstrate at least a 4-fold increase, at least a 4.5-fold increase, orat least a 5-fold increase in binding capacity upon dual-agonistactivation

Also provided is a method of treating a bleeding disorder in a subjectin need thereof, the method comprising administering a therapeuticallyeffective amount of an alternative therapy to a subject having plateletsthat demonstrate a 1.6-fold increase in binding capacity or less or a1.3-fold increase in binding capacity or less upon dual-agonistactivation (e.g., activation with thrombin and convulxin) compared torFVIIa binding capacity of non-activated platelets from the subject. Forexample, in various embodiments, the subject has platelets thatdemonstrate a 1.0-fold increase or less upon dual-agonist activationcompared to rFVIIa binding capacity of non-activated platelets from thesubject. rFVIIa capacity increase may be determined by calculating theratio of median fluorescence increase (MFI) of dual-agonist activatedplatelets versus non-activated platelets from the subject. rFVIIacapacity increase also may be determined by calculating MFI ofnon-activated platelets, activating the platelets using dual agonists,calculating the MFI of activated platelets, and determining the ratio ofthe MFI values.

In one aspect, the invention includes a subpopulation of plateletsdescribed herein (e.g., a subpopulation of platelets have at least a4-fold higher binding capacity to rFVIIa as compared to the bindingcapacity to rFVIIa of other platelets in the platelet population fromwhich the subpopulation was derived). Also included is a plateletpopulation comprising platelets having a higher binding capacity torFVIIa as compared to the binding capacity to rFVIIa of platelets in acontrol platelet population. Also provided is a platelet population thatdemonstrates at least a 3.5-fold increase in binding capacity upondual-agonist activation compared to rFVIIa binding capacity ofnon-activated platelets obtained from same the subject, and a plateletpopulation that demonstrates at least a 15-fold higher rFVIIa relativefluorescence after exposure to 100 nM rFVIIa, as described herein. Thesubpopulation of platelets or the platelet population is optionally inthe form of a pharmaceutical composition.

In one embodiment, the pharmaceutical composition is used in a method oftreating a bleeding disorder, such as hemophilia (e.g., hemophilia A orhemophilia B) or a non-hemophilia bleeding disorder, in subject. Invarious aspects, the method comprises administering to a subject atherapeutically effective amount of a platelet population containingcomprising the subpopulation of platelets described herein, e.g., asubpopulation of platelets have at least a 4-fold higher bindingcapacity to rFVIIa as compared to the binding capacity to rFVIIa ofother platelets in the platelet population). Optionally, thesubpopulation comprises at least 0.5%, at least 1%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, at least 13%, at least14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19%, at least 20%, at least 21%, at least 22%, at least 23%, at least24%, at least 25%, at least 26%, at least 27%, at least 28%, at least29%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95% or 100% of the plateletpopulation administered to the subject. The invention furthercontemplates the subpopulation of platelets for use in treating ableeding disorder, or for use in preparing a medicament for treating ableeding disorder.

The invention also includes a method of treating a bleeding disorder ina subject in need thereof, wherein the method comprises administering tothe subject a therapeutically effective amount of a platelet population,wherein the platelets in the platelet population have a higher bindingcapacity to rFVIIa as compared to the binding capacity to rFVIIa ofplatelets in a control platelet population. The invention furthercontemplates the population of platelets for use in treating a bleedingdisorder, or for use in preparing a medicament for treating a bleedingdisorder.

Hemophilia and non-hemophilia disorders are described above. The subjectis, in various embodiments, a mammal, such as a human or a rodent (e.g.,mouse or rat), cow, pig, sheep, goat, cat, horse, dog, or other speciesof animal used as livestock or kept as pets. In one aspect, the subjecthas been diagnosed with a bleeding disorder, is predisposed to (or is atrisk of) developing a bleeding disorder, or is suspected to have ableeding disorder. In various embodiments, the subject is being treatedfor a bleeding disorder before being treated according to the methods ofthe invention. In other embodiments, the subject is not being treatedfor a bleeding disorder before being treated according to the methods ofthe invention.

In certain embodiments, the subject is a human and a platelet populationor a subpopulation of platelets according to the methods describedherein are human cells. The platelets are activated (coated or notcoated) or not activated. The platelets may comprise a predeterminedpercentage of activated or non-activated platelets and/or apredetermined percentage of coated or non-coated (e.g., activated,non-coated) platelets. For instance, the platelet subpopulation orpopulation administered to a patient optionally comprises at least 50%,at least 60%, at least 70%, at least 80%, at least 90% or at least 99%of a desired platelet type. It will be understood that the plateletpopulation or subpopulation administered need not be absolutelyhomogenous (i.e., 100% of a particular platelet type); less than 100%(e.g., 99%, 98%, or 97% or less) also is contemplated herein.

In some embodiments, a platelet population or a subpopulation ofplatelets according to the methods described herein can be supplied inthe form of a pharmaceutical composition, comprising pharmaceuticalcarrier (e.g., an isotonic excipient) prepared under sufficientlysterile conditions for human administration and selected on the basis ofthe chosen route of administration and standard pharmaceutical practice.Techniques and formulations generally can be found in Remmington'sPharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20^(th) Ed.,2000), the entire disclosure of which is herein incorporated byreference. For general principles in medicinal formulation of platelets,see Sweeney et al., 1995, Quality of Platelet Concentrates,Immunological Investigations, 24(1&2), 353-370; and Stroncek D. F. andRebulla P., 2007, Platelet Transfusions, The Lancet, 370(9585), 427-438.According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theplatelet population or subpopulation of platelets according to theinvention can be used. Supplementary active compounds can also beincorporated into the compositions. Lyophilized forms of compositions(or platelet populations or subpopulations) are also included.Compositions of the invention are characterized as being at leaststerile and pyrogen-free. The compositions include formulations forhuman and veterinary use.

Choice of the excipient and any accompanying elements of the compositioncomprising a platelet population or a subpopulation of plateletsaccording to the methods described herein will be adapted in accordancewith the route and device used for administration. Examples of routes ofadministration include parenteral, e.g., intravenous or intraarterial,administration. Solutions or suspensions used for parenteral applicationcan include one or more of the following components: a sterile diluentsuch as water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; and buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, containers, or blood bags madeof glass or plastic.

In some embodiments, a composition comprising a platelet population or asubpopulation of platelets according to the methods described herein canalso comprise, or be accompanied with, one or more other ingredientsthat facilitate the delivery or functional mobilization of the plateletpopulation or a subpopulation of platelets according to the methodsdescribed herein. Suitable ingredients include, for example, citratephosphate dextrose (CPD) solution, plasma solutes, glucose, phosphatebased buffering systems, bicarbonate based buffering systems, fattyacids, amino acids, sodium acetate, or other ingredients that supportthe storage and delivery of a platelet population or a subpopulation ofplatelets according to the methods described herein. In anotherembodiment, the composition may comprise autologous blood plasma orblood plasma (for additional information, see Sweeney et al., 1995).

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. Liquid formulations can comprise one or morephysiologically compatible buffers, such as citrate phosphate dextrosesolution (see e.g., Sweeney et al., 1995). For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, apharmaceutically acceptable polyol like glycerol, propylene glycol,liquid polyetheylene glycol, and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it can be useful to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofinjectable compositions can be brought about by incorporating an agentwhich delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theplatelet population or a subpopulation of platelets in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating aplatelet population or a subpopulation of platelets of the inventioninto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated herein.

Examples of diluents and/or carriers and/or other additives that may beused include, but are not limited to, water, glycols, oils, alcohols,aqueous solvents, organic solvents, DMSO, saline solutions,physiological buffer solutions, peptide carriers, starches, sugars,preservatives, antioxidants, coloring agents, pH buffering agents,granulating agents, lubricants, binders, disintegrating agents,emulsifiers, binders, excipients, extenders, glidants, solubilizers,stabilizers, surface active agents, suspending agents, tonicity agents,viscosity-altering agents, carboxymethyl cellulose, crystallinecellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate. The combination of diluentsand/or carriers and/or other additives used can be varied taking intoaccount the nature of the active agents used (for example the solubilityand stability of the active agents), the route of delivery (e.g. oral,parenteral, etc.), whether the agents are to be delivered over anextended period (such as from a controlled-release capsule), whether theagents are to be co-administered with other agents, and various otherfactors. One of skill in the art will readily be able to formulate thecomposition for the desired use without undue experimentation.

For parenteral administration (i.e., administration by through a routeother than the alimentary canal), a platelet population or subpopulationof platelets of the invention may be combined with a sterile aqueoussolution that is isotonic with the blood of the subject. Such aformulation may be prepared by dissolving the active ingredient in watercontaining physiologically-compatible substances, such as sodiumchloride, glycine and the like, and having a buffered pH compatible withphysiological conditions, so as to produce an aqueous solution, thenrendering the solution sterile. The formulation may be presented in unitor multi-dose containers, such as, but not limited to, containers orblood bags. The formulation may be delivered by injection, infusion, orother means known in the art.

In some embodiments, a platelet population or subpopulation of plateletsof the invention is provided in unit dose form such as a single-doseinjection or infusion vial.

A platelet population or a subpopulation of platelets according to theinvention is administered by any means available for use in conjunctionwith pharmaceuticals, either as individual therapeutic activeingredients or in a combination of therapeutic active ingredients. Invarious embodiments, a platelet population or a subpopulation ofplatelets according to the methods described herein is formulated andadministered to reduce the symptoms associated with a bleeding disorderby any means that allow the platelets to exert their effect on thesubject in vivo, e.g., administered to any suitable location allowingplatelet binding to rFVIIa. In various aspects, a platelet population ora subpopulation of platelets according to the methods described hereinis administered systemically. A platelet population or a subpopulationof platelets of the invention may be administered parenterally, e.g., byintravascular, intravenous, or intraarterial delivery. Delivery may beeffected by injection, infusion, or catheter delivery. In oneembodiment, a platelet population or a subpopulation of platelets of theinvention is administered to the subject directly to the vascular systemvia catheter inserted into a vein of the subject. Methods ofadministering a composition to a subject are further described inSweeney et al., 1995 and Stroncek et al., 2007.

Administration of a platelet population or a subpopulation of plateletsis not restricted to a single route, but may encompass administration bymultiple routes. Multiple administrations (whether by the same route ordifferent routes of administration) may be sequential or concurrent.Other modes of application by multiple routes will be apparent to one ofskill in the art.

In certain embodiments, a platelet population or a subpopulation ofplatelets according to the methods described herein can be administeredand dosed in accordance with good medical practice, taking into accountthe clinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to one of skill in the art, such as, butnot limited to, a medical practitioner. A platelet population or asubpopulation of platelets according to the methods described herein maybe administered to a subject in a therapeutically effective amount totreat a bleeding disorder. A “therapeutically effective amount,” forpurposes herein, is thus determined by such considerations as are knownin the art. The amount can be effective to achieve improvement,including, but not limited to, improved bleeding time, improved clottingtime, improved prothrombin time, improved partial thromboplastin time,improved activated clotting time, or improvement or elimination ofsymptoms and other indicators as are selected as appropriate measures bythose skilled in the art. For additional information, see e.g., TheAbsite Review by Steven M. Fiser, Lippincott Williams and Wilkins 2004.

A therapeutically effective amount of a platelet population or asubpopulation of platelets that treats a bleeding disorder can dependupon a number of factors known to those of ordinary skill in the art.The dose(s) of a platelet population or a subpopulation of plateletsaccording to the methods described herein can vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which aplatelet population or a subpopulation of platelets according to themethods described herein, is to be administered, if applicable, and theeffect which the practitioner desires the platelet population or asubpopulation of platelets according to the invention to have upon thetarget of interest. These amounts can be readily determined by one ofskill in the art. These amounts include, for example, number ofplatelets per kilogram (kg) of subject weight, such as about 1×10⁹cells/10 kg, about 1×10¹⁰ cells/10 kg, about 1×10¹¹ cells/10 kg, about1×10¹² cells/10 kg, or between about 1×10⁹ cells/10 kg to 1×10¹²cells/10 kg, 1×10⁹ cells/10 kg to 1×10¹⁰ cells/10 kg, 1×10¹⁰ cells/10 kgto 1×10¹¹ cells/10 kg, or to 1×10¹¹ cells/10 kg to 1×10¹² cells/10 kg,or any range in between. These amounts also include a unit dose ofplatelets, for example, at least 1×10⁹ cells, 1×10¹⁰ cells, 1×10¹¹cells, 1×10¹² cells, or 1×10¹³ cells, or more. For additionalinformation, see Stroncek et al., 2007. Any of the therapeuticapplications described herein can be applied to any subject in need ofsuch therapy, including, for example, a mammal, such as a human.

Appropriate dosing regimens can also be determined by one of skill inthe art without undue experimentation in order to determine, forexample, whether to administer the agent in one single dose or inmultiple doses, and in the case of multiple doses, to determine aneffective interval between doses. For example, in one aspect, a plateletpopulation or a subpopulation of platelets described herein isadministered to the subject once (e.g., as a single injection orinfusion). Alternatively, a platelet population or a subpopulation ofplatelets according to the methods described herein is administered onceor twice daily to a subject in need thereof for a period of from, e.g.,about two to about twenty-eight days, or from about seven to about tendays. A platelet population or a subpopulation of platelets according tothe methods described herein can also be administered once or twicedaily to a subject over the course of a year, wherein the once or twicedaily administration occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12times per year.

In certain embodiments, a platelet population or a subpopulation ofplatelets according to the methods described herein is stored for laterinjection/infusion. In some embodiments, a platelet population or asubpopulation of platelets according to the methods described herein canbe stored for up to 5 days, up to one week, or up to two weeks. Foradditional information, see Stroncek et al., 2007.

Therapy dose and duration will depend on a variety of factors, such asthe disease type, patient age, therapeutic index of the drugs, patientweight, and tolerance of toxicity. Initial dose levels will be selectedbased on their ability to achieve ambient concentrations shown to beeffective in in vivo models, and in clinical trials. The skilledclinician using standard pharmacological approaches can determine thedose of a particular therapeutic and duration of therapy for aparticular patient in view of the above stated factors. The response totreatment can be monitored by analysis of coagulation measures, and oneof skill in the art, such as a clinician, will adjust the dose andduration of therapy based on the response to treatment revealed by thesemeasurements.

In various embodiments, a platelet population or a subpopulation ofplatelets according to the methods described herein are administeredalone (e.g., as a single agent) or in combination with one or more otheragents (e.g., therapeutic agents). Suitable additional agents include,but are not limited to, cells, tissue, tissue fragments, biologicallyactive or inert compounds, and small molecules.

In various embodiments, a platelet population or subpopulation ofplatelets of the invention are used in combination with other agentsthat are used for the treatment or prevention of a bleeding disorders,such as clotting factors (e.g., Factor VIIa (rFVIIa), Factor VIII(FVIII), and Factor IX (FIX)). Examples of agents suitable for use inconnection with the platelet population or subpopulation include, butare not limited to, recombinant Factor VIIa, such as Novoseven®, FEIBA,BeneFix® (recombinant Factor IX), Kogenate® FS (recombinant FactorVIII), Recombinate (recombinant Factor VIII), Advate® (recombinantFactor VIII), Helixate® FS (recombinant Factor VIII), Koāte®-DVI(recombinant Factor VIII), Stimate® (desmopressin acetate), DDAVP®(desmopressin acetate), Bebulin (Factor IX Complex), Hemofil M® (humanfactor VIII), cryoprecipitated antihaemophilic factor (AHF), freshfrozen plasma (FFP), recombinant porcine FVIII, recombinant FV variants,recombinant FVIIa variants, recombinant FXa variants, FXIII,prothrombin, a mix of coagulation factors, antibodies mimicking FVIII,peptides mimicking FVIII, compounds mimicking FVIII, peptide inhibitorsof TFPI, antibody inhibitors of TFPI, compounds inhibiting TFPI,compounds inhibiting anti-coagulant proteins, agents resulting inreduced expression of anti-coagulant proteins (e.g., compounds thatreduce expression or antisense oligonucleotides that reduce expressionof an anti-coagulant protein), and any therapy for treating a bleedingdisorder that does not comprise rFVIIa as a single agent, as determinedby a person of skill in the art.

In various embodiments, a platelet population or subpopulation ofplatelets of the invention are used in combination with agents that arenot used for the treatment or prevention of bleeding disorders, such as(but not limited to) additives intended to, for example, enhance thedelivery, efficacy, tolerability, or function of the platelet populationor a subpopulation of platelets.

A platelet population or subpopulation of platelets of the invention andother agents may be administered to the subject at the same time or atdifferent times. For example, in one embodiment, a platelet populationor subpopulation of platelets of the invention is delivered to a subjectas part of the same pharmaceutical composition or formulation containingone or more additional agents. Alternatively, one or more other agentsare administered to the subject in one or more separate compositions orformulations. A platelet population or subpopulation of platelets of theinvention and one or more other agents are optionally administeredwithin minutes, hours, days, weeks, or months of each other, for exampleas part of the overall treatment regimen of a subject. A plateletpopulation or subpopulation of platelets of the invention is optionallyadministered prior to the administration of other agents. In variousembodiments, a platelet population or subpopulation of platelets of theinvention is administered subsequent to the administration of otheragents.

A platelet population or subpopulation of platelets of the invention mayalso be used in combination with surgical or other interventionaltreatment regimens used for the treatment of bleeding disorders. In someembodiments, a platelet population or subpopulation of platelets of theinvention is used as an adjuvant therapy. In other embodiments, aplatelet population or subpopulation of platelets of the invention isused in combination with an adjuvant therapy.

In another aspect, the invention provides a method of determiningwhether a subject is a candidate for treatment with rFVIIa, the methodcomprising (a) obtaining a sample of platelets derived from the subject;(b) incubating a platelet population from the sample with rFVIIa; and(c) detecting whether the platelet population contains a subpopulationof platelets having at least a 4-fold higher binding capacity to rFVIIa,as compared to the binding capacity to rFVIIa of the other platelets inthe platelet population. In one aspect, this candidate is a candidatefor treatment with rFVIIa if the subject does not have an unacceptablerisk of thrombosis, and if the platelet population contains asubpopulation of platelets having at least a 4-fold higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of theother platelets in the platelet population. In various embodiments, thecandidate is a candidate for treatment if the subpopulation representsat least 2%, at least 3%, at least 4%, at least 5%, at least 10%, atleast 15%, or at least 20% of the platelet population. In anotheraspect, the subject is a candidate for treatment with rFVIIa if thesubject has an unacceptable risk of thrombosis, and if the plateletpopulation does not contain a subpopulation of platelets having at leasta 4-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of the other platelets in the platelet population. Invarious embodiments, the platelet population sampled contains less than5%, less than 3%, less than 2%, or less than 1% of the subpopulation.Methods of characterizing FVIIa binding, features of the platelets ofthe platelet population and subpopulation, composition of the plateletpopulation and subpopulation (e.g., percentage of platelets with highbinding capacity), subjects, biological samples, and bleeding disordersare described herein and applicable to the method.

EXAMPLE

This example is provided below to facilitate a more completeunderstanding of the present invention. However, the scope of theinvention is not limited to specific embodiments disclosed in thisexample, which are for purposes of illustration only.

Example 1 Detection of High Donor Variation in Binding of RecombinantActivated Factor VII (rFVIIa) to Human Platelets, and of a SubpopulationBinding High Amounts of rFVIIa

The variation of the rFVIIa binding capacity of platelets in plateletconcentrates from 21 healthy donors, and the occurrence and frequency ofa platelet subpopulation capable of binding high amounts of rFVIIa, aredescribed herein.

Methods

Platelets are identified by staining for CD61 and P-selectin (CD62P) andforward/side scatter gating to discriminate their activation status.Coated platelets can be identified by using a fluorochrome conjugatedanti-fibrinogen antibody. rFVIIa bound to activated or non-activatedplatelets is quantified using a DyLight488-labeled anti human FVIIaantibody. Fresh platelet concentrates are adjusted to 40,000 plateletsper μl in 20 mM HEPES and 150 mM NaCl containing buffer adjusted topH7.35. Platelets were diluted with 9 volumes buffer containing 5.5 mMCaCl₂, 0.11 μg/mL convulxin (a collagen receptor GPVI agonist) and 5.5nM thrombin, if activation was required, and incubated with rFVIIa(BAX817 or Novoseven) at final concentrations from 50 to 2000 nM for7-15 min at 37° C. and 300 rpm. Then, platelets were fixed with 3.8%final concentration of paraformaldehyde contained in the same buffer and5 mM CaCl₂. Platelets were filtered through a 50 μm mesh filter andblocked for 10 min at room temperature by adding 5% fetal bovine serum.Then, platelets are centrifuged for 10 min at 4° C. and 700 g. Forstaining of rFVIIa bound to the platelet surface, a rabbit anti humanFVIIa polyclonal Dylight 488 antibody conjugate (Affinity BioReagentsorder #PA1-100250) was used at a final concentration of 20 μg/mL. After15 min incubation at room temperature in the dark, platelets arecounterstained by adding mouse monoclonal antibodies against CD61 (BDorder #22458; conjugated to PerCP, 1.2 μg/ml final concentration) andCD62P (BD order #01732; P-selectin, conjugated to PE, final dilution1:5) and incubated for 15 min at room temperature in the dark.Fibrinogen can be co-stained using a Dylight633 conjugated sheepanti-human fibrinogen antibody (Affinity Biologicals order #SAFG-AP; at10 μg/mL). Then, wash buffer is added (same buffer w/o FBS), mixed andcentrifuged for 10 min at 4° C. and 700 g. Samples are resuspended forFACS analysis. Controls include platelets without rFVIIa, and a mouseIgG1 isotype control conjugated to PerCP (BD order #) at 1.2 μg/mL finalconcentration, and samples stained with each antibody alone for propercompensation of fluorescence spillover. Median fluorescence intensitiesof rFVIIa bound to all platelets and to sub-populations varying inrFVIIa binding were determined by flow cytometry. K_(D) values werecalculated using SigmaPlot v12.0.

Results

FACS raw data typical for one experiment are shown in FIG. 1, where 100nM rFVIIa have been added without platelet activation and incubated for7 min, and the same sample and handling steps with dual agonistactivation in FIG. 2.

CD61 and CD62P Signals on Non-Activated and Activated Platelets

The CD61 signal versus the isotype control for gating of platelets isshown in FIG. 3 for activated and non-activated platelets. Activationincreases the CD61 signal approximately two-fold. Varying incubationtimes of 7, 10 and 15 min do not have an influence on the CD61 signal.

Activated and non activated platelets as depicted in FIG. 4 stained withan anti-CD62P antibody-fluorochrome conjugate show that there is noinfluence of the activation time of 7, 10 and 15 min on the CD62Pquantity on activated platelets. In FIG. 5, the CD62P signal is shown asan indicator for platelet activation. The CD62P signal was compared tonon-activated platelet background in N7 or BAX817 formulation buffer. Itwas shown that the CD62P signal intensity does not depend on rFVIIaconcentration. Without being bound by theory, a higher degree ofactivation can be caused by BAX817 without activators. It is noted thatN7 did not lead to higher activation.

Dose Dependent Binding of rFVIIa to Platelets

In FIG. 6, the titration of rFVIIa on platelets is shown. The medianfluorescence values of rFVIIa bound to activated and non-activatedplatelets (CD61-positive population) are shown. Binding of BAX817 wassomewhat higher than N7; ratios of binding to activated or non-activatedplatelets were similar. Binding of both rFVIIa preparations was showndose dependent on activated or non activated platelets until saturationwas reached. Without being bound by theory, a similar plateletinteraction for BAX817 and Novoseven can be observed at rFVII aconcentrations of 100 nM.

Detection of a rFVIIa High Binding Subpopulation

An additional, strongly positive FVII—fluorescent population wasdetected, which is shifting to the left closer to the major peak withlonger incubation times (7, 10, 15 min) (mean and median fluorescencebecomes lower over time). The percentage of the high FVIIa binding andthe major platelet population stayed equal. Two NovoSeven lots weretested and show similar behavior.

Histogram overlay of the FVII DyLight488 staining for different samplesas indicated. Population is gated by forward scatter versus side scatterand CD61 (FIG. 7).

In FIG. 8, platelet histograms and dotplots of non-activated andactivated platelets from a typical donor are shown. Dotplots of therFVIIa against the CD62P signal (FIG. 9) did not show a difference inthe amounts of CD62P on the main and the rFVIIa high binding population.In FIG. 9 co-staining of rFVIIa and fibrinogen on platelets from onedonor having the high-rFVIIa binding subpopulation is shown. The amountof fibrinogen exposed by the high rFVIIa binding platelets could not bedistinguished from fibrinogen exposed by the major platelet populationbefore and after activation. For resting platelets, a 2-fold increase inCD62P expression was observed for high binding platelet sub-populations,which was low compared to that in CD62P expression observed uponplatelet activation (˜40-fold).

In FIG. 10, the platelet population is homogenous in terms of fibrinogenand CD62P exposure, but not rFVIIa binding (100 nM rFVIIa added). Thehigh level of fibrinogen indicates that all platelets are “coated”.Without being bound by theory, the subpopulation of platelets can beconsidered small.

In FIG. 11, the bright side-population that was detected was shown to belosing signal with time. This is shown also in FIG. 14.

TABLE 1 Mean and median fluorescence of FITC pos 1 (=major) populationand the FITC pos 2 (=high rFVIIa binding) population after 7, 10 and 15min incubation times FITC pos 1 FITC pos 2 Median Mean CV % of MedianMean CV % of Samples FITC FITC FITC parent FITC FITC FITC parentplatelets_5 25.41 28.13 52.17 81.05% 271.45 302.17 52.65 14.97% activ 7min Novoseven # 1 platelets_8 24.87 27.50 51.22 81.49% 173.83 196.8856.24 15.41% activ 10 min Novoseven # 1 platelets_11 24.91 27.46 50.4080.96% 93.77 107.98 57.62 15.39% activ 15 min Novoseven # 1 platelets_622.36 24.97 52.65 81.55% 328.93 363.61 48.93 13.72% activ 7 minNovoseven # 2 platelets_9 21.45 24.20 54.40 80.87% 199.54 229.02 60.4214.05% activ 10 min Novoseven # 2 platelets_12 21.96 24.47 52.33 77.55%89.98 108.76 70.21 16.47% activ 15 min Novoseven # 2

Dot plot of FVII versus CD62 of Novoseven lot#1 for different plateletsamples as indicated. Platelets were activated and incubated withNovoseven for 7 minutes (top), 10 minutes (middle), or 15 minutes(bottom) (FIG. 12).

Dot plot of FVII versus CD62 of Novoseven lot#2 for different plateletsamples as indicated. Platelets were activated and incubated withNovoseven for 7 minutes (top), 10 minutes (middle), or 15 minutes(bottom) (FIG. 13).

Detection of High Donor Variation in rFVIIa Platelet Binding, andCharacterization of Donors Regarding the High Binding Subpopulation

Using FACS technology, binding of rFVIIa to platelet concentrates of 21healthy donors was quantified, based on the assumption that the amountof rFVIIa bound to per cell correlates with the measured fluorescenceintensity. In Tables 2 (non-activated) and 3 (double-activatedplatelets), all donors are listed with protocol addresses, K_(D) valuesif calculable, relative median fluorescence intensities of the rFVIIasignals of all platelets compared to the controls without rFVIIa, and offrequencies and relative median fluorescence intensities of high bindingpopulations compared to the major rFVIIa binding population. rFVIIabound concentration dependently to non-activated and dual agonistactivated platelets. On activated and non-activated platelets, bindingwas saturated at rFVIIa concentrations between 200 and 1200 nM. Medianfluorescence intensity increase at 100 nM rFVIIa ranged from 4- to33-fold for non-activated (mean±standard deviation: 14±7.5-fold) andfrom 5- to 37-fold for activated platelets (mean±standard deviation:15±9.5-fold) compared with controls without rFVIIa. When assessing thetotal platelet population, binding constants for activated plateletsranged from 29 to 1025 nM (mean±standard deviation 392±355 nM; n=6), andfrom 56 to 357 nM (mean±standard deviation 184±88; n=10) fornon-activated platelets.

A platelet sub-population consisting of at least 2% was detected in 7 of21 non-activated platelet donor concentrates, and in 15 of 21 after dualactivation. In one donor, this population was visible only withinresting platelets, in 9 donors only within activated platelets, and in 6donors within resting and activated platelets. Up to 24% of totalplatelets were characterized by high capacity of rFVIIa binding inplatelet concentrates from donors positive for this sub-population.

Regarding all donors described, this subpopulation bound on average32-fold more rFVIIa than the main platelet population whennon-activated, and on average 6-fold more within activated platelets.The rFVIIa high binding capacity subpopulation formation did notcorrelate with platelet activation or “coated” platelet formation, sinceall dual agonist activated platelets had similar distribution of CD62Pand fibrinogen expression following activation.

A substantial inter-individual variation in binding of rFVIIa to restingand activated platelets was observed among a group of 21 donors. In somedonor platelet concentrates, sub-populations of platelets witha >10-fold increase in rFVIIa binding, consisting of up to >20% of totalplatelets after activation, were identified.

This finding supports prediction of therapy outcomes or thrombogenicrisks with rFVIIa or other bypassing therapies when studying a patient'splatelet population, but also serves as means to improve hemophiliainhibitor bypassing therapy e.g. by recruiting platelet donorspossessing the ability to bind high amounts of rFVIIa and isolatingtheir platelets for transfusion. The invention solves the problem of alack of understanding about this platelet population which is needed tocarry out successful hemophilia therapy, off-label use of rFVIIa, orgene therapy when targeting platelets in an effort to treat bleedingdisorders. Prior to this invention, there was no explanation whyplatelets bind more or less rFVIIa, but with this invention includingfunctional and phenotypic characterization and identification ofcorrelating factors, one induce/suppress high binding of rFVIIa toplatelets in a patient.

TABLE 2 Percentages of the high rFVIIa binding sub-population, andratios of fluorescence of the high binding versus the main plateletpopulation for all rFVIIa concentrations measured from all donors;non-activated platelets.

Values for populations <2% are in italic with grey fillings. “N/A” = notapplicable. “MFI” = median fluorescence intensity.

TABLE 3 Percentages of the high rFVIIa binding sub-population, andratios of fluorescence of the high binding versus the main plateletpopulation for all rFVIIa concentrations measured from all donors; dualagonist-activated platelets.

Values for populations <2% are italic with grey fillings. “N/A” = notapplicable. “MFI” = mean fluorescence intensity.

TABLE 4 K_(D) estimations of high rFVIIa binding platelet subpopulationscompared to the major populations (populations > 2% were included); cutoffs: K_(D) R² > 0.90. Only activated platelets from some donorsfulfilled these criteria. Donor protocol K_(D) [nM] address high rFVIIabinding population Major population JK069 685 JK066 200 109 JK062 554935 AH163 354 AH155 413

The methods described above were employed to characterize binding ofrFVIIa to platelet concentrates of additional healthy donors and usingdifferent lots of rFVIIa. Analysis of 19 non-activated platelet samplesresulted in a mean apparent K_(D) (calculated from ligand bindingcurves) of 276 nM (±200 SD) for rFVIIa with a range of 56-897 nM.Analysis of 18 dual-agonist activated platelet samples resulted in amean apparent K_(D) of 312 nM (±194 SD) for rFVIIa with a range of36-632 nM. A statistical overview of studies of 37 donors is provided inFIG. 16A and FIG. 16B. When the high rFVIIa binding population was below2% total platelets in an experiment, the X-fold MFI of the highpopulation versus the major platelet population was excluded. Thehigh-binding subpopulation of non-activated platelets bound about 6-foldto about 52-fold more rFVIIa than the rest of the population, with amean increase of about 22-fold. The range of percentage of FVIIahigh-binding platelets in a sample was 0% to about 8.4%; the meanpercentage was about 1.6% of the platelet population. The high-bindingsubpopulation of dual-activated platelets bound about 3-fold to about20-fold more rFVIIa than the rest of the population of dual-activatedplatelets, with a mean increase of about 7-fold. The highest percentageof rFVIIa high-binding platelets in a sample was 48%; the mean was about4.5% of the activated platelet population. rFVIIa binding todual-agonist activated platelets was compared to rFVIIa binding bynon-activated platelets in samples. The mean increase in rFVIIa bindingof dual-agonist activated platelets to non-activated platelets(MFI_(activated)/MFI_(non-activated)) ranged from 1.03 to 5.20 with anaverage of 2.16±0.96. A boxplot diagram illustrating the distribution ofrFVIIa binding capacity increases observed in platelet samples isprovided in FIG. 19.

The distribution of a rFVIIa high-binding subpopulation among activatedvs. non-activated platelets also was determined. Platelet samples wereexposed to 100 nM or 50-4000 nM rFVIIa. All donors were classified basedon the existence of a high rFVIIa binding subpopulation of at least 2%of all platelets and further subdivided based on the existence of a highrFVIIa binding population present in activated only, non-activatedplatelets only, or both. Representative results are provided in Table 5:

100 nM 50-4000 nM rFVIIa rFVIIa Numbers of donors tested 35 100% 37 100%High rFVIIa binding subpopulation in: non-activated platelets only 1  3%3  8% activated platelets only 15 43% 15 41% activated and non-activatedplatelets 11 31% 14 38% No high rFVIIa binding subpopulation 8 23% 5 14%

Boxplot diagrams illustrating the distribution of overall capacity ofnon-stimulated and dual-agonist activated platelets to bind rFVIIa upontreatment with 100 nM rFVIIa are set forth in FIGS. 20A and 20B. 5% ofdonors do not bind more than 1.2-fold rFVIIa after dual-agonisttreatment than before stimulation, 10% do not bind more than 1.3-fold(lower whisker of FIG. 19), 20% do not bind more than 1.5-fold, 25% donot bind more than 1.6-fold (lower box border of FIG. 19), 40% do notbind more than 1.8-fold, 50% do not bind more than 1.9-fold (median lineof FIG. 19), 60% do not bind more than 2.1-fold, 75% do not bind morethan 2.2-fold (upper box border of FIG. 19), 80% do not bind more than2.4-fold, 90% do not bind more than 3.7-fold (upper whisker of FIG. 19),and 95% do not bind more than 5.1-fold rFVIIa after dual-agonistactivation compared to non-activated counterparts. With respect to thedata underlying FIG. 20A, platelets from 5% donors do not have more than5.3-fold higher rFVIIa relative fluorescence (MFI than the buffercontrol), if non-stimulated and exposed to 100 nM rFVIIa, 10% donors donot have more than 6.3-fold higher rFVIIa relative fluorescence (lowerwhisker), 20% donors do not have more than 8.0-fold higher rFVIIarelative fluorescence, 25% donors do not have more than 8.0-fold higherrFVIIa relative fluorescence (lower box border), 40% donors do not havemore than 8.5-fold higher rFVIIa relative fluorescence, 50% donors donot have more than 11.0-fold higher rFVIIa relative fluorescence, 60%donors do not have more than 11.0-fold higher rFVIIa relativefluorescence, 75% donors do not have more than 15.5-fold higher rFVIIarelative fluorescence (upper box border), 80% donors do not have morethan 18.7-fold higher rFVIIa relative fluorescence, 90% donors do nothave more than 25.2-fold higher rFVIIa relative fluorescence (upperwhisker), and 95% donors do not have more than 32.8-fold higher rFVIIarelative fluorescence, if non-stimulated and exposed to 100 nM rFVIIa.With respect to the data underlying FIG. 20B, platelets from 5% donorsdo not have more than 5.4-fold higher rFVIIa relative fluorescence (MFIthan the buffer control), if dual-agonist activated and exposed to 100nM rFVIIa, 10% donors do not have more than 6.6-fold higher rFVIIarelative fluorescence (lower whisker), 20% donors do not have more than8.2-fold higher rFVIIa relative fluorescence, 25% donors do not havemore than 9.8-fold higher rFVIIa relative fluorescence (lower boxborder), 40% donors do not have more than 11.5-fold higher rFVIIarelative fluorescence, 50% donors do not have more than 13.0-fold higherrFVIIa relative fluorescence, 60% donors do not have more than 14.0-foldhigher rFVIIa relative fluorescence, 75% donors do not have more than20.6-fold higher rFVIIa relative fluorescence (upper box border), 80%donors do not have more than 22.0-fold higher rFVIIa relativefluorescence, 90% donors do not have more than 29.5-fold higher rFVIIarelative fluorescence (upper whisker), and 95% donors do not have morethan 34.2-fold higher rFVIIa relative fluorescence, if stimulated bydual agonists and exposed to 100 nM rFVIIa.

Activated platelets that demonstrated high rFVIIa binding was notlimited to coated platelets; the subpopulation of high rFVIIa binding,activated platelets also included non-coated platelets. FIGS. 17A and17B describe population sizes and MFIs for rFVIIa and fibrinogen fornon-activated and dual-agonist activated platelet samples from 5 donors.The amount of rFVIIa bound and fibrinogen exposure definedsubpopulations. The percentages of each population of all platelets andMFIs of the FVII/FVIIa and fibrinogen stainings are provided. If norFVIIa was added, quadrants (Q) show the following: Q1: FVIIa negative,fibrinogen positive; Q2: FVIIa positive, fibrinogen positive; Q3: FVIIapositive, fibrinogen negative; Q4: FVIIa negative, fibrinogen negative.For all other samples, quadrants correspond to Q1: FVIIa main,fibrinogen positive; Q2: FVIIa high, fibrinogen positive; Q3: FVIIahigh, fibrinogen negative; Q4: FVIIa main, fibrinogen negative. Thedual-activated platelet data serves the basis for the bubble diagrams inFIG. 18A-18B. As illustrated, high rFVIIa binding activity wasidentified in both coated (Q2, high fibrinogen staining) and non-coated(Q3, low fibrinogen staining) platelets.

While this invention has been described with an emphasis upon variousembodiments, it will be obvious to those of ordinary skill in the artthat variations of the methods may be used and that it is intended thatthe invention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the invention as defined bythe following claims.

What is claimed is:
 1. A method of treating a bleeding disorder in asubject in need thereof, the method comprising administering atherapeutically effective amount of rFVIIa to the subject, wherein a) asample of platelets derived from the subject was obtained; b) a plateletpopulation from the sample was incubated with rFVIIa; and c) asubpopulation of platelets having at least a 4-fold higher bindingcapacity to rFVIIa, as compared to the binding capacity to rFVIIa of theother platelets in the platelet population, was detected in the plateletpopulation.
 2. The method of claim 1, wherein the bleeding disorder is anon-hemophilia bleeding disorder, and (d) the subject does not have anunacceptable risk of thrombosis.
 3. A method of treating a bleedingdisorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of an alternativetherapy to the subject, wherein a) a sample of platelets derived fromthe subject was obtained; b) a platelet population from the sample wasincubated with rFVIIa; and c) a subpopulation of platelets having atleast a 4-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation, was not detected in the platelet population.
 4. The methodof claim 3, wherein the alternative therapy is BeneFix®, Kogenate® FS,Recombinate, Advate®, Helixate® FS, Koāte®-DVI, Stimate®, DDAVP®,Bebulin, Hemofil M®, cryoprecipitated antihaemophilic factor (AHF),fresh frozen plasma (FFP), Prothrombin Complex Concentrate, or activatedProthrombin Complex Concentrate.
 5. The method of claim 4, wherein theactivated Prothrombin Complex Concentrate is FEIBA.
 6. The method ofclaim 3, wherein the alternative therapy is recombinant porcine FVIII,recombinant FV variants, recombinant FVIIa variants, recombinant FXavariants, FXIII, prothrombin, fibrinogen, a mix of coagulation factors,antibodies mimicking FVIII, peptides mimicking FVIII, compoundsmimicking FVIII, peptide inhibitors of TFPI, antibody inhibitors ofTFPI, compounds inhibiting TFPI, or compounds inhibiting anti-coagulantproteins.
 7. The method of any one of claims 1-6, wherein the detectionis by flow cytometry.
 8. The method of any one of claims 1-7, whereinthe sample of platelets is obtained from a blood sample or a serumsample from the subject.
 9. The method of any one of claims 1-8, whereinthe sample of platelets is a fresh sample, a concentrate, a preservedsample, a rehydrated lyophilized sample, or a frozen sample.
 10. Amethod of treating a bleeding disorder in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of a platelet population containing a subpopulation ofplatelets having at least a 4-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population.
 11. The method of any one of claims 1 and 3-10,wherein the bleeding disorder is hemophilia.
 12. The method of claim 11,wherein the hemophilia is hemophilia A or hemophilia B.
 13. The methodof claim 12, wherein the hemophilia A is congenital hemophilia A withinhibitors or acquired hemophilia A with inhibitory auto antibodies toFVIII, and the hemophilia B is congenital hemophilia B with inhibitorsor acquired hemophilia B with inhibitory auto antibodies to FIX.
 14. Themethod of any one of claims 1 and 3-18, wherein the bleeding disorder isa non-hemophilia bleeding disorder.
 15. The method of claim 2 or 14,wherein the non-hemophilia bleeding disorder is selected from the groupconsisting of blood loss from trauma, FVII deficiency, FV deficiency, FXdeficiency, FXI deficiency, FXIII deficiency, fibrinogen deficiency,prothrombin deficiency, dilutional coagulopathy, thrombocytopenia, bloodloss from high-risk surgeries, intracerebral hemorrhage, von Willebranddisease, and von Willebrand disease with inhibitors to von Willebrandfactor.
 16. A method of determining whether a subject is a candidate fortreatment with rFVIIa, the method comprising a) obtaining a sample ofplatelets derived from the subject; b) incubating a platelet populationfrom the sample with rFVIIa; c) detecting whether the plateletpopulation contains a subpopulation of platelets having at least a4-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of the other platelets in the platelet population,wherein a subject is a candidate for treatment with rFVIIa if (i) thesubject does not have an unacceptable risk of thrombosis, and theplatelet population contains a subpopulation of platelets having atleast a 4-fold higher binding capacity to rFVIIa, as compared to thebinding capacity to rFVIIa of the other platelets in the plateletpopulation; or (ii) the subject has an unacceptable risk of thrombosis,and the platelet population does not contain a subpopulation ofplatelets having at least a 4-fold higher binding capacity to rFVIIa, ascompared to the binding capacity to rFVIIa of the other platelets in theplatelet population.
 17. The method of claim 16, wherein the sample ofplatelets is obtained from a blood sample or a serum sample from thesubject.
 18. The method of claim 16 or claim 17, wherein the sample ofplatelets is a fresh sample, a concentrate, a preserved sample, arehydrated lyophilized sample, or a frozen sample.
 19. The method of anyone of claims 16-18, wherein the subject has a bleeding disorder. 20.The method of claim 19, wherein the bleeding disorder is hemophilia,blood loss from trauma, FVII deficiency, FV deficiency, FX deficiency,FXI deficiency, FXIII deficiency, fibrinogen deficiency, prothrombindeficiency, dilutional coagulopathy, thrombocytopenia, blood loss fromhigh-risk surgeries, intracerebral hemorrhage, von Willebrand disease orvon Willebrand disease with inhibitors to von Willebrand factor.
 21. Themethod of any one of claims 16-20, wherein the detection is by flowcytometry.
 22. The method of any one of claims 1-21, wherein theplatelets in the subpopulation of platelets are activated.
 23. Themethod of any one of claims 1-21, wherein the platelets in thesubpopulation of platelets are non-activated.
 24. The method of any oneof claims 1-21, wherein the platelets in the subpopulation of plateletsare coated.
 25. The method of any one of claims 1-24, wherein theplatelet population contains a subpopulation of platelets having about a6-fold higher, about a 20-fold higher, about a 30-fold higher, or abouta 40-fold higher binding capacity to rFVIIa, as compared to the bindingcapacity to rFVIIa of the other platelets in the platelet population.26. The method of any one of claims 1-25, wherein the subject is ahuman.
 27. A method of treating a bleeding disorder in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a platelet population, wherein theplatelets in the platelet population demonstrate at least a 15-foldhigher rFVIIa relative fluorescence after exposure to 100 nM rFVIIa. 28.The method of claim 27, wherein the platelet population comprisesnon-activated platelets demonstrating at least a 25-fold higher rFVIIarelative fluorescence after exposure to 100 nM rFVIIa.
 29. The method ofclaim 27, wherein the platelet population comprises activated plateletsdemonstrating at least a 20-fold higher rFVIIa relative fluorescenceafter exposure to 100 nM rFVIIa.
 30. The method of any one of claim 27,wherein the platelet population comprises activated plateletsdemonstrating at least a 29-fold higher rFVIIa relative fluorescenceafter exposure 100 nM to rFVIIa.
 31. The method of claim 27, wherein theplatelets in the platelet population have a rFVIIa binding constant ofabout 50 to 400 nM or about 25 to 1100 nM.
 32. A method of treating ableeding disorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of rFVIIa to thesubject, wherein a) a sample of platelets derived from the subject wasobtained; b) a non-activated platelet population from the sample wasincubated with rFVIIa; and c) platelets demonstrating at least a 15-foldhigher, optionally at least a 25-fold higher, rFVIIa relativefluorescence after exposure to 100 nM rFVIIa were detected in theplatelet population.
 33. A method of treating a bleeding disorder in asubject in need thereof, the method comprising administering atherapeutically effective amount of rFVIIa to the subject, wherein a) asample of platelets derived from the subject was obtained; b) anactivated platelet population from the sample was incubated with rFVIIa;and c) platelets demonstrating at least a 20-fold higher, optionally atleast a 30-fold higher, rFVIIa relative fluorescence after exposure to100 nM rFVIIa were detected in the platelet population.
 34. A method oftreating a bleeding disorder in a subject in need thereof, the methodcomprising administering a therapeutically effective amount of analternative therapy to the subject, wherein a) a sample of plateletsderived from the subject was obtained; b) a non-activated plateletpopulation from the sample was incubated with rFVIIa; and c) plateletsdemonstrating a 8-fold higher rFVIIa relative fluorescence or less,optionally a 5-fold higher rFVIIa relative fluorescence or less, afterexposure to 100 nM rFVIIa were detected in the platelet population. 35.A method of treating a bleeding disorder in a subject in need thereof,the method comprising administering a therapeutically effective amountof an alternative therapy to the subject, wherein a) a sample ofplatelets derived from the subject was obtained; b) an activatedplatelet population from the sample was incubated with rFVIIa; and c)platelets demonstrating a 9-fold higher rFVIIa relative fluorescence orless, optionally a 5-fold higher rFVIIa relative fluorescence or less,after exposure to 100 nM rFVIIa were detected in the plateletpopulation.
 36. The method of any one of claims 32-35, wherein thedetection is by flow cytometry.
 37. A method of treating a bleedingdisorder in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of rFVIIa to a subjecthaving platelets that demonstrate at least a 3.5-fold increase inbinding capacity upon dual-agonist activation compared to rFVIIa bindingcapacity of non-activated platelets from the subject.
 38. A method oftreating a bleeding disorder in a subject in need thereof, the methodcomprising administering a therapeutically effective amount of analternative therapy to a subject having platelets that demonstrate a1.3-fold increase in binding capacity or less upon dual-agonistactivation compared to rFVIIa binding capacity of non-activatedplatelets from the subject.
 39. The method of any one of claims 32-38,wherein the bleeding disorder is hemophilia.
 40. The method of claim 39,wherein the hemophilia is hemophilia A or hemophilia B.
 41. The methodof claim 40, wherein the hemophilia A is congenital hemophilia A withinhibitors or acquired hemophilia A with inhibitory auto antibodies toFVIII, and the hemophilia B is congenital hemophilia B with inhibitorsor acquired hemophilia B with inhibitory auto antibodies to FIX.
 42. Themethod of any one of claims 32-38, wherein the bleeding disorder is anon-hemophilia bleeding disorder.
 43. The method of claim 42, whereinthe non-hemophilia bleeding disorder is blood loss from trauma, FVIIdeficiency, FV deficiency, FX deficiency, FXI deficiency, FXIIIdeficiency, fibrinogen deficiency, prothrombin deficiency, dilutionalcoagulopathy, thrombocytopenia, blood loss from high-risk surgeries,intracerebral hemorrhage, von Willebrand disease or von Willebranddisease with inhibitors to von Willebrand factor.
 44. The method of anyone of claims 34, 35, and 38, wherein the alternative therapy isBeneFix®, Octanate®, Rixubis®, Kogenate® FS, Recombinate, Advate®,Helixate® FS, Koāte®-DVI, Stimate®, DDAVP®, Bebulin, Hemofil M®,cryoprecipitated antihaemophilic factor (AHF), or fresh frozen plasma(FFP), Prothrombin Complex Concentrate, or activated Prothrombin ComplexConcentrate.
 45. The method of claim 44, wherein the activatedProthrombin Complex Concentrate is FEIBA.
 46. The method of any one ofclaims 34, 35, and 38, wherein the alternative therapy is recombinantporcine FVIII, recombinant FV variants, recombinant FVIIa variants,recombinant FXa variants, FXIII, prothrombin, fibrinogen, a mix ofcoagulation factors, antibodies mimicking FVIII, peptides mimickingFVIII, compounds mimicking FVIII, peptide inhibitors of TFPI, antibodyinhibitors of TFPI, compounds inhibiting TFPI or compounds inhibitinganti-coagulant proteins or reducing expression of anti-coagulantproteins.
 47. The method of any one of claims 32-46, wherein the sampleof platelets is obtained from a blood sample or a serum sample from thesubject.
 48. The method of any one of claims 32-47, wherein the sampleof platelets is a fresh sample, a concentrate, a preserved sample, arehydrated lyophilized sample, or a frozen sample.
 49. The method of anyone of claims 27-48, wherein the subject is a human.
 50. The method ofclaim 3, wherein the alternative therapy is Octanate® or Rixubis®. 51.The method of any one of claims 1-22, wherein the platelets in thesubpopulation of platelets are not coated.
 52. A subpopulation ofplatelets isolated from a platelet population, wherein the platelets inthe subpopulation of platelets have at least a 4-fold higher bindingcapacity to rFVIIa as compared to the binding capacity to rFVIIa of theother platelets in the platelet population.
 53. The subpopulation ofplatelets of claim 52, wherein the higher binding capacity to rFVIIa isdetermined by flow cytometry.
 54. The subpopulation of platelets ofclaim 52 or 53, wherein the platelets in the subpopulation of plateletsare activated.
 55. The subpopulation of platelets of claim 52 or 53,wherein the platelets in the subpopulation of platelets arenon-activated.
 56. The subpopulation of platelets of claim 52 or 53,wherein the platelets in the subpopulation of platelets are coated. 57.The subpopulation of platelets of any one of claims 52-54, wherein theplatelets in the subpopulation of platelets are not coated.
 58. Thesubpopulation of platelets of any one of claims 52-57, wherein theplatelets in the subpopulation of platelets have about a 6-fold higher,about a 20-fold higher, about a 30-fold higher, or about a 40-foldhigher binding capacity to rFVIIa, as compared to the binding capacityto rFVIIa of the other platelets in the platelet population.
 59. Thesubpopulation of platelets of any one of claims 52-58, wherein thesubpopulation of platelets is supplied in the form of a pharmaceuticalcomposition.
 60. The subpopulation of platelets of claim 59, wherein thepharmaceutical composition is for systemic administration to a subjectin need thereof.
 61. The subpopulation of platelets of claim 59 or 60,wherein the pharmaceutical composition is for treating a bleedingdisorder, wherein the bleeding disorder is optionally hemophilia, bloodloss from trauma, FVII deficiency, FV deficiency, FX deficiency, FXIdeficiency, FXIII deficiency, fibrinogen deficiency, prothrombindeficiency, dilutional coagulopathy, thrombocytopenia, blood loss fromhigh-risk surgeries, intracerebral hemorrhage, von Willebrand disease orvon Willebrand disease with inhibitors to von Willebrand factor.
 62. Thesubpopulation of platelets of any one of claims 52-61, wherein thesubject is a human.
 63. A platelet population containing a subpopulationof platelets having at least a 4-fold higher binding capacity to rFVIIaas compared to the binding capacity to rFVIIa of the other platelets inthe platelet population for use in treating a bleeding disorder.
 64. Aplatelet population comprising platelets having a higher bindingcapacity to rFVIIa as compared to the binding capacity to rFVIIa ofplatelets in a control platelet population for use in treating ableeding disorder.